## 4 Discussion
In studies of breast cancer cell lines, it was observed that increasing concentrations of drugs may trigger many epigenetic mechanisms leading to the development of resistance to chemotherapeutic drugs. [PERSON] et al. showed that the concentration of docetaxel that induced MDR caused hypermethylation of the main \(ABCB1\) promoter and switched the transcription to the upstream promoter. Further increasing the drug concentration caused a higher mRNA level and preceded a regional chromosome 7 amplification containing, i.a., a gene encoding P-gp [36]. Similarly, \(ABCB1\) expression can be significantly upregulated in lung cancer cells resistant to a cisplatin analogue [37] and paclitaxel [38]. This is accompanied by an increase in the amount of mRNA for surrounding genes (including \(RUNDC3B\) or \(ABCB4\)), which is proof of the multiplication of the entire \(ABCB1\) amplicon [38]. Similar observations apply to neuroblastoma cancer cell lines as wells as leukemia and myeloma cells [45; 46]. The activation of this mechanism probably depends on the types, doses, and concentrations of the chemotherapeutic drugs that are used in experiments with cancer cell lines. Although it lacks a TATA box, the \(ABCB1\) downstream promoter has an initiator (Inr) motif, an inverted CCAAT box, and two GC boxes. Furthermore, binding motifs for a large number of transcription factors are located in this region. It also contains a CpG island; therefore, the majority of scientific reports concern this \(ABCB1\) promoter, as it is responsible for the epigenetic regulation of the gene's function. In a study by [PERSON] et al. conducted on 19 cancer cell lines (lung, colon, breast, cervix, ovary, prostate, osteosarcoma, multiple myeloma, and leukemia), the differences in the methylation status of the main \(ABCB1\) promoter were observed. In over half of these cell lines, high methylation of the investigated CpG islands was observed, and this was connected with the lack of an encoded mRNA/protein. For lung cancer, three cell lines (HCC827, NCI-H1703, and SW 1573) indicated a high methylation status, while the promoter regions of two other lines (A549 and NCI-H520) were methylated rather heterogeneously [47]. The presence of unique DNA methylation patterns specific to cancer cell line types was also confirmed in other studies, e.g., for lung and prostate cancer cell lines [48; 49]. In our study, the analysis of the DNA methylation status of the \(ABCB1\) gene was performed on clinical lung cancer sample data derived from the TCGA database. As was presented in the results, we found that the promoter of \(ABCB1\) was hypo-methylated in both normal tissue and lung cancer tissue (beta-value range: 0.022-0.138). Nevertheless, in both LUAD and LUSC cancer cells, the level of promoter methylation was significantly higher compared to normal lung cells. These observations were consistent with other lung cancer studies [37; 48], confirming that the methylation of the promoter region is more frequent in cancers than in non-cancerous tissues. Similarly, [PERSON] et al. detected methylation in the majority of investigated lung cancer samples, but in this study, all normal lung tissue samples also showed promoter methylation [50]. In this case, it cannot be ruled out that the patients with inflammatory pseudotumors, considered as a control group, had an altered DNA methylation pattern due to an inflammatory process. Our findings are also in line with related studies in other types of cancer. For example, the difference in the frequency of \(ABCB1\) promoter methylation was significant between prostate cancer and benign gland hypertrophy samples. Although the presence of methylated and unmethylated CpG sites was found in both groups, methylated gene regions and weak expression of \(ABCB1\) predominated in the cancer cells [51]. Moreover, hypermethylation in the CpG sites of this gene appears to be quite common in various untreated primary tumors [52].