The molecular basis of allelic loss in lung cancer has been suggested to be DNA adducts. Allelic losses are eas­ily found by analyzing polymorphic tandem repeat se­quences in the genome. In these studies chromosomal loci normally have two different polymorphic alleles. The loss of both of these is called homozygous deletion and causes silencing of the gene, the loss of one of them is defined as LOH. Allelic loss may result in functional loss only when the retained allele is deleted or silenced through methyla­tion – the Knudson effect.

      In lung cancer, LOH studies have established that there are many regions of allelic loss in smoking-damaged epi­thelium but rarely does this loss lead to any effect on gene expression [45]. Allelic loss appears early in the period of smoking exposure, but it does not appear in non smokers. Results from LOH analyses showed multiple sites of genetic alternations in 1p, 4p, 4q, 5q, 6q, 8p, 9p, 9q, 10p, 10q, 13q, 18q, 21q.32 [45]. Common sites of deletions in lung cancers are at 3p14.2 FHIT (in 100% of SCLC and in 53% of NSCLC), 3p25-26 (in 100% of SCLC and in 50% of NSCLC), 10q (PTEN), 9p21 (p16) [28]. Deletions of 3p are present in 86% of dysplasia tissues and in 76% of hyperplasia [46].

      Loss of heterozygosity at locus 9p13 is an early event being seen in the normal appearing epithelium of smokers and in preinvasive lesions and reversible after smoking cessation [47]. A study of the relation between smoking duration and mechanisms of gene silencing showed that LOH of 9p13 and homozygous deletion is linearly corre­lated with smoking, and is determined by the innate sus­ceptibility and smoking exposure [48]. Individuals with 9p21 LOH had better survival rates than those with homo­zygous deletion [48].

      Telomerase Activity. Telomeres DNA consist of re­petitive duplex TTAGGG tracts. Telomere-specific pro­teins bind directly to the single- and the double-strand regions and form a complex that provides a protective cap over the ends of the chromosomes that protect them from degradation, recombination, and end-joining reactions [49]. Telomeres ensure complete replication and contribute to the functional organization of the chromosomes in the nucleus. The limited proliferative capacity of human cells is caused by lack of detectable telomeres or of sufficient telomerase activity. This leads to telomere shortening with each replication and to eventual loss of the telomere cap.

      Telomere shortening in the absence of telomerase is the fundamental mechanism by which cells regulate the number of divisions [50]. The finding that the telomerase activity is repressed in normal tissue but is present in 85-90% of human cancers has led to an increasing interest in the role of telomeres and telomerase activity in carcino­genesis. Telomerase is the enzyme that adds TTAGGG nucleotide repeats onto the telomere ends of DNA to com­pensate for the loss during replication [51]. Somatic cells lack telomerase and stop dividing when the telomere ends of at least some chromosomes are shortened to a critical level.

      Telomerase activity was compared in preneoplastic, non cancerous and in SCLC and NSCLC tissues; 50% of preneoplastic lesions were telomerase positive [52]. In SCLC telomerase was nearly always present and in NSCLC it was observed in 75% [53].

      The results of immunohistochemistry correlated with the protein distribution in tissue sections. In most normal epithelial cells no h TRET expression was detected but in squamous and adenocarcinoma the protein was found in the nucleus of almost all neoplastic cells and correlated with the level of telomerase activity (Table 1) [54]. In general, telomerase activity is more precise marker in can­cer cells than hTRET mRNA which occurs as several splice variants.

      Epigenetic Changes. These are molecular changes that do not alter the base sequence of DNA but may result in altered gene expression. They contrast with genetic changes (mutations) that alter the DNA base sequence [55]. Two major changes influence epigenetic transcrip­tional control, DNA methylation and deacytylation of his­tone proteins, of which the former is commonly involved in lung carcinogenesis.

      DNA Methylation. DNA methylation was identified early as a hallmark of epigenetic silencing of TSG [56]. Hypermethylation of short CpG-rich regions (known as CpG islands) was found in malignant cells. These islands exist in the promoter regions of many transcribable genes and are generally unmethylated in normal cells [57]. The hypermethylation of the CpG islands is observed in almost all cancers but the genes altered are specific for each tumor type [58].

      Difference in methylation status of cancer-emerging genes was observed between different types of lung cancer by Toyooka et al. [59]. They established that neuroendo­crine tumors that include SCLC and carcinoids had a high­er frequency of methylation of RASSF1A (TSG located on 3p.21), compared to NSCLC tumors (p <0.0001), while the frequency of methylation of p16, APC, CDH13 were high­er in NCSLC than in SCLC (p <0.0001). Within the NSCLC group, squamous cancers had the highest fre­quency of p16 methylation, while adenocarcinomas had the highest frequency of APC and CDH13 methylation. The higher prevalence of p16 methylation in squamous cell carcinoma was confirmed by Kim et al. [44]. The APC methylation was associated with non squamous histology. Recent researches have thrown light upon the role of PAX5 b gene which indirectly regulates CD19 that negatively controls cell growth [60].

      So the genes of interest as potential methylation mark­ers are p16, APC, RASSF1A, RAR-b, DAPK, PAX5, CDH13. It was established that the methylation of one or more of these genes is associated with a poor prognosis [61]. Thus, patients with a bad prognosis might then be selected based on their DNA methylation profile.

      In lung cancer smoking has been reported to be respon­sible for the methylation of a log of genes [62]. In 514 cases of NSCLC and 84 corresponding non malignant lung tissues, they found insignificant gender-related differences, while the methylation index was definitely higher in smok­ers than in non smokers. It has been established that the methylation of the p16 gene is often found in the carcino­genesis induced by chronic inflammation and oxidative stress in models of rats exposed to carbon black and diesel exhaust. So it is reasonable to believe that in lung cancer the DNA methylation might be related to chronic inflam­mation and oxidative stress induced by smoking [63].

      Belinsky et al. [64] first determine that p16 methyla­tion in an animal model of lung cancer and in human squa­mous cell carcinoma is a very early event. In humans, p16 methylation is found in 75% of carcinoma in situ lesions adjacent to squamous cell carcinoma and the frequency of p16 methylation increases with the progression of the dis­ease. Lamy et al. [65] also demonstrated methylation of p16 in 19% of pre-invasive lesions of high-risk individuals and the frequency increased with the histological grade. The determinants of aberrant methylation are not well characterized. Gilliand et al. [66] hypothesized that the functional polymorphism of NADPH, glutathione-S-trans­feraseP1 and M1 myeloperoxidase and XRCC1 genes are associated with p16 and MGMT methylation in sputum.

      DNA Adducts. DNA adducts are modifications of DNA that indicate exposure to carcinogens. They are phys­ical complexes between DNA and the reactive metabolites of tobacco smoke and industrial pollutants. DNA adduct is an early abnormality that is not seen at the chromosomal level. Adducts can interfere with DNA repair and mitosis can lead to mitotic recombination or partial chromosomal loss. Gene methylation, mutation and further mitotic re­combination may occur and result to a high level of aneu­ploidy that is present in lung cancer. Thus adducts, by causing copy number changes, can directly alter regulation of transcription of TSG or of oncogenes [67]. As the level of adducts in tumor tissue and in blood lymphocytes have been associated with lung cancer [68], and because these levels correspond to daily cigarette consumption and do not reverse after smoking cessation [69], it is suggested that DNA adducts should serve as potential biomarkers of risk for lung cancer.

      In conclusion, the development of lung cancer seems to be a continuous and complex process in which a number of genes and the effect of exogenic factors are involved. Genetic polymorphisms of the enzymes responsible for the metabolism of cigarette smoke, and polymorphisms of genes involved in DNA repair processes, determine an individual’s sensitivity towards the carcinogens in the cigarette smoke. This explains why only 10-15% of smok­ers develop lung cancer.

Tissue	Telomerase Activity (%)	hTRET mRNA (%)
Non cancerous	15.3	6.7
Pre cancerous	52.1	37.1
Small cell cancer	92.3	75.0
Non small cell cancer	73.4	51.8