Chromosomal Instability Of Human Tracheal Epithelial Cells Beas-2b Induced By Coal Tar Pitch Smoke Extracts

BackgroundCoal Tar Pitch (CTP), one of chemical carcinogens discovered long ago, could cause occupational cancer, but its pathogenesis remains unclear. In this study, the effect of chromosomal instability (CIN) during the evolved process of carcinogenesis was studied by analyzing the chromosome variation of malignant transformation in BEAS-2B cells induced by coal tar pitch smoke extracts.Chromosome instability is characterized by an elevated rate of gains or losses of complete chromosomes or segments of chromosomes in the process of cell mitosis. Chromosome instability is one of the most notable character of malignant cell which is presented in most tumors.The phenomenon of chromosome instability includes the changes in chromosome number and structure. The former, which is also called aneuploidy, refers to whole chromosome lost or/and gained. The latter involves chromosome deletion, inversion, translocation, duplication, ring chromosome, dicentric chromosome, etc.Chromosome instability is involved in every stage of cell cycle in mitosis, especially including four aspects, such as abnormal sister chromatid condensation and segregation, abnormal centrosome, defects in spindle checkpoint function and telomere abnormalities.1. Abnormal condensation and segregation of sister chromatidAfter DNA replication, two chromatids are linked in the ring by cohesion to pair sister chromatid. When Sccl subunit is hydrolyzed by Separase, the chromatid would be released easily. Most of the time in the cell cycle, the hydrolytic activity of Separase is inhibited resulting from combining with Securin. In the middle and late stages of the cell cycle, Securin is degraded and Separase can be released to hydrolyze cohesion, then sister chromatids are separated.2. Weakened spindle checkpoint function If the genes of Mad or Bub mutate or decrease the expression, and cells still could divide ignoring the damage of the spindle, abnormal karyotypes will be formed. When the incorrect kinetochore is detected, the checkpoint associated protein, named Mad2, is activated to inhibit the APC/C activity. Securin can not be degraded and sister chromatids can not be separated. Spindle checkpoint function in tumor cell is weaker than that in normal cell, and accompanied by high-frequency of CIN.3. Abnormal centrosomeThere is a positive correlation between centrosome abnormalities and CIN(aneuploidy), which is prevalent in solid tumors. Over-expression of centrosome is a common phenomenon especially in lung cancer, which is accompanied with abnormal expression of morphology, size and number. The replication process of centrosome is regulated strictly and CDK2/CyclinE complex plays a very important role. P53 can regulate centrosome replication by inhibiting CDK2/CyclinE in the dependent P53/P21 pathway manner. Inactivation or reduced expression of P53 and over-expression of CyclinE could cause centrosome amplification and CIN. There is positive correlation between P53 and CIN/centrosome amplification. Centrosome abnormalities contribute to CIN and have a profound impact to the occurrence of cancer.4. Telomeres, telomerase abnormalitiesIn most of the tumor cells, telomere shortens and telomerase activity increases to maintain the immortalized cells. When telomere shortens to a certain degree, telomere ends could integrate and "bridging-fusion-breakage" cycle is formed, then karyotype of tumor cell is appeared and tumorigenesis is initiated. TRF1 is mainly responsible for negative regulation of telomere length and regulation of telomere extension. TRF2 is mainly responsible for the protection of chromosome ends. If TRF2 lost, chromosome ends will lose the protection, then telomere 3'ends lost and chromosome terminal-terminal will be fused. POT1 is concerned with the regulation of telomerase activity.Karyotype analysis demonstrate that there is a complex karyotype in almost all lung cancer. Triploid, tetraploid, chromosome translocation, chromosome deletion and heterochromatin have been showed in about 70%-80% of lung cancer. As mentioned above, the change of both the number and the structure of Chromosome can cause the change of gene composition.But it is still controversial what role of CIN has played in the process of the occurrence, development and evolution of tumor.Therefore, in this study, immortalized human bronchial epithelial cells BEAS-2B was induced by middle-temperature coal tar pitch smoke extracts, to establish BEAS-2B malignant transformation model. During the process, the chromosome variation, the cell transformation and the relationship were observed on every stage of cell malignant transformation, and the mechanism was analyzed on the four aspects: abnormal condension and segregation of sister chromatid, weakened spindle checkpoint function, centrosome abnormalities and telomeres/telomerase abnormalities, to explore the mechanism of lung cancer induced by CTP and to provide the theoretical basis for preventing occupational tumor exposed to CTP.ObjectiveTo analyze components of coal tar pitch and establish the model of malignant transformation of human bronchial epithelial cells BEAS-2B, then observe the relationship between chromosomal instability and malignant transformation in cell phase.Four aspects, including abnormal sister chromosome condensation and segregation, spindle checkpoint, abnormal centrosome and telomeres/telomerase abnormal were studied to analyze the mechanism of chromosomal instability, approache mechanism of lung cancer caused by coal tar pitch.Materials and Methods1. Experimental material:Medium temperature coal tar pitch:It obtained from Anyang Iron and Steel Company coking plant, was crushed into fine powder (φ10μm～20μm). The temperature was controlled at about 400℃. As it produces smoke automatically, smoke dust were collected, then extracted by dichloromethane, dissolved in DMSO and storaged at room temperature for use. Cell lines:immortalized human bronchial epithelial cells BEAS-2B cells. Nude mice:24 BALB/C nude mice, female,5-6 weeks, weight 10-20g, SPF grade, purchased from Hunan Slack King of Laboratory Animal Co., Ltd..2. Methods2.1 Analysis of coal tar pitch smoke extracts composition and cytotoxicity on BEAS-2B cellsCoal tar pitch smoke extracts solution in DMSO was analyzed and identified by gas chromatography/mass spectrometer. The components were identified by automatic search through NIST mass spectrometry database, and relative quantification was calculated by the peak area normalization.The cytotoxicity of BEAS-2B cells caused by coal tar pitch smoke extracts was tested by MTT cell proliferation assay and the LC50 was calculated.2.2 BEAS-2B cell malignant transformation induced by coal tar pitch smoke extracts and observation of chromosomal instabilityBEAS-2B cells were induced by coal tar pitch smoke extract at 2.0mg/L Morphological changes were observed in the passage and transformation; the cell anchorage independent growth was detected in every 10 generations, then the tumor formation in nude mice was verified, chromosome karyotype of cells was observed by traditional methods. When cells showed morphological changes, cell cycle was detected by flowcytometry and chromosomes subtle structural variation were observed by M-FISH.The detection of DNA damage by coal tar pitch:the cell was exposed for 24h with the induced dose (2.0mg/l), and DNA damage on BEAS-2B cells caused by coal tar pitch smoke extracts was observed by single cell gel electrophoresis.2.3 Mechanism of chromosomal instability induced by coal tar pitch smoke extractsCentrosome changes of 10th,20th. and 30th generation induced cells were detected by indirect immunofluorescence method. Telomere length was detected by fluorescence quantitative PCR, and telomerase activity was measured by fluorescence quantitative TRAP assay. The gene expression levels of mRNA were assessed by real-time quantitative RT-PCR, the protein expression variation were determined by cell culture overslip of immunohistochemical methods.3. Statistical analysisData were expressed as mean±S.D and processed by SPSS 12.0. Differences among groups were examined for statistical significance using one-way analysis of variance, differences between two groups were examined by LSD test. The test level a= 0.05 (bilateral).Results1. The components of coal tar pitch smoke extract and acute toxicity on BEAS-2B cells1.1 Components analysisThere were totally 38 compounds identified in coal tar pitch smoke extracts by GC/MS, which were mainly polycyclic aromatic hydrocarbons, accounting for 87.91%, and the rest was monocyclic aromatic hydrocarbon, heterocyclic and alkene compounds.1.2 Cytotoxicity testWith the increasing of the concentration of coal tar pitch, the inhibition on BEAS-2B cells strengthened gradually. The curve was symmetrical S-curve and the LC50 was 8.64mg/L.2. Coal tar pitch smoke extracts induced BEAS-2B cells transformation and chromosome instabilityBEAS-2B cells were induced to transform with induction dose (2.0 mg/L). After 30 generations cultures, there was a malignant transformation on BEAS-2B cells.2.1 Morphological changeThere was no obvious morphological change on cells of the 10th generation. The morphology of some cells were abnormal in the 20th generation in induced group and the cells grew exuberant; in the 30th generation, the cells of the induced group were scrambled without rules, the size of cells were different significantly, and some cells lost contact inhibition, appeared clad growth phenomenon and special-shaped nuclear fission and pathologic phase.2.2 Changes in cell cycle It showed that the proportion of G1 phase cells decreased and that of G2/M phase cells increased significantly by flow cytometry in the 30th generation induced cells. This indicates that induced cells are in proliferation exuberant state and grew more quickly than the cells in control group.2.3 Soft agar coloning-formation assay and tumor formation in nude miceThere was rare coloning formation in the 10th generation induced cells, about 0.4‰. The coloning formation rate increased to 5.93‰in the 20th generation induced cells, which was higher than those in the other two groups. The coloning formation rate was 21.50‰in the 30th generation induced cells, much higher than that in normal control group and DMSO group, respectively. The colonies were in disorder morphology and no contact inhibition in coal tar pitch group.38d after inoculation the 30th cells induced by coal tar pitch in nude mice, the tumors' size and weight were significantly higher than those in the other 3 groups, the differences were statistically significant (P<0.05). HE staining showed the structure of tumor was disorder, and there were abnormal-shaped nuclear and the formation of small blood vessels in the 30th cells induced by coal tar pitch. This indicates that the 30th cells induced by coal tar pitch have been already malignantly transformed.2.4 Chromosome instabilityIn the 10th generation induced cells, the proportion of diploid karyotype decreased obviously, and the proportion of aneuploid, particuly sub-diploid and hyperdiploid was increased markedly, especially the diploid and super diploid ratio increased considerably. Using columnχ2 contingency table test analysis, cell chromosome number among three groups was statistically significant difference (P< 0.05). Afterχ2 segmentation analysis, compared to the normal control group and DMSO group, induced group was statistically significant difference (P<0.05), while difference between the normal control group and DMSO group was no significant (P>0.05).In the 20th generation induced cells, the proportion of diploid karyotype(17%) decreased significantly, and sub-diploid (19%), hyperdiploid(62%) and polyploid increased further. In the 30th generation induced cells, the proportion of diploid karyotype significantly decreased(7%), and there were a large number of aneuploid cells, including the sub-diploid (15%) and hyperdiploid (78%).The M-FISH results showed that the karyotypes of BEAS-2B cells itself had been in an unstable state. Although nearly diploid was main karyotype, there was a large number of cross-exchange in chromosome structure. The karyotypes of cells induced by coal tar pitch were very unstable, and there were a large number of polyploid and sub-diploidy, chromosomes shift, cross-exchange and deletion. The fine chromosomes structures of karyotype was varied each other.2.5 The DNA damage effect of coal tar pitch on BEAS-2B cellsBEAS-2B cells was exposed to coal tar pitch with the induction dose (2.0mg/L) for 24h, single-cell gel electrophoresis showed that Tail length, tail moment, comet length and Olive tail moment were significantly increased (P<0.05), compared to the blank control group and DMSO group. That indicates that the coal tar pitch itself can act directly on BEAS-2B cells to cause DNA damage, resulting in genetic toxicity.3 The chromosomal instability mechanism of BEAS-2B cells induced by coal tar pitch smoke extracts3.1 The effects of coal tar pitch smoke extracts on the sister chromatid condension & segregation related gene of BEAS-2B cellsIn 30th induced cells, the mRNA expression level of SMC1 gene was no statistically significant difference compared to the BEAS-2B group and DMSO group (P> 0.05); SMC3 and Separase gene mRNA expression level were increased and Securin gene mRNA expression level was reduced, the differences compared to the other two groups were significantly (P<0.05), while the difference between other two groups was no significant (P> 0.05).The results of immunohistochemistry showed:In 30th induced cells, the protein expression of SMC1 gene was no statistically significant differences compared to the BEAS-2B group and DMSO group (P> 0.05); SMC3 and Separase protein expression were increased and Securin protein expression was decreased, the differences compared to the other two control groups were significant (P<0.05), while the difference between other two control groups was no significant (P> 0.05).3.2 The effects of coal tar pitch smoke extracts on the spindle checkpoint-related protein of BEAS-2B cellsIn 30th induced cells, the mRNA expression level of Mad2, Bub land APC gene was decreased, the differences compared to the normal control group and DMSO group were significant (P<0.05), while the difference between other two control groups was no significant (P> 0.05). the pretoin expression of Mad2, Bub land APC gene was decreased, the differences compared to the normal control group and DMSO group were significant (P<0.05), while the difference between other two control groups was no significant (P> 0.05).3.3 The effects of coal tar pitch smoke extracts on centrosome of BEAS-2B cells3.3.1 The variation of centrosome:In the 10th generation induced cell, we found a lot of centrosome abnormalities of the number and morphological, the total abnormality rate was 2.79%, higher than the other two groups. However, the differences among the 3 groups was no statistically significant (P> 0.05).In the 20th generation induced cell, the total abnormal centrosome was 6.56%, mainly in the abnormalities of the number (3.41%), significantly higher than the control group and DMSO group(P<0.05), but the differences between the control group and DMSO group was no statistically significant (P> 0.05).In the 30th generation induced cell, there is 3 types abnormal centrosome, including the abnormalities of number (12.70%), the abnormalities of shape (5.69%), the abnormalities of number and shape (4.00%). The cell proportion of the total anomalies (22.39%) was significantly increased(P<0.05), and the other two groups difference was no significant (P> 0.05).3.3.2 The mRNA level variation of P53,P21 and CyclinE geneIn the 10th generation induced cell, the mRNA levels of P53, P21 and CyclinE was no statistically significant differences (P> 0.05). The mRNA levels of P53 and P21 was decreased and the CyclinE was increased in 20th and 30th cells in coal tar pitch group compared to the BEAS-2B group and DMSO group, the differences were statistically significant (P<0.05).3.3.3 The protein expression variation of P53,P21 and CyclinE geneIn the 10th generation induced cell, the protein expression of P53, P21 and CyclinE was no statistically significant differences (P> 0.05). The protein expression of P53 and P21 was decreased and CyclinE was increased in 20th and 30th cells in coal tar pitch group compared to the BEAS-2B group and DMSO group, the differences were statistically significant (P<0.05).3.4 The effects of coal tar pitch smoke extracts on telomere, telomerase of BEAS-2B cells3.4.1 The variation of telomere length and telomerase activityCompared to the BEAS-2B group and DMSO group, the relative length of telomere DNA shortening, telomerase activity increased in 20th and 30th generation coal tar pitch cells, the differences were statistically significant (P<0.01). The 10th generation cell, the normal control group, DMSO group and the coal tar pitch group compared, the indexes in the groups was no statistically significant (P> 0.05).3.4.2 The mRNA level variation of POT1, TRF1 and TRF2 geneThe mRNA levels of POT1 and TRF1 gene was decreased and the TRF2 gene was increased in 20th and 30th cells in coal tar pitch group compared to the BEAS-2B group and DMSO group, the differences were statistically significant (P＜0.05). Each index of the 10th generation cells in the 3 groups showed no significant difference (P> 0.05).3.4.3 The protein expression variation of POT 1, TRF1 and TRF2 geneThe protein expression of POT 1 and TRF1 gene was decreased and the TRF2 gene was increased in 20th and 30th cells in coal tar pitch group compared to the BEAS-2B group and DMSO group, the differences were statistically significant (P <0.05 or P<0.01). Each index of the 10th generation cells in the 3 groups showed no significant difference (P> 0.05).ConclusionsThe components of coal tar pitch smoke extracts are complex, which mainly are polycyclic aromatic hydrocarbons. It has genetic toxicity effects on BEAS-2B cells, and can induce BEAS-2B cells chromosomal instability and malignant transformation in vitro.Chromosomal abnormalities occurred in early stage of cells malignant transformation before the changes in cell morphology and malignant function in the model of malignant transformation cell in BEAS-2B cells induced by coal tar pitch smoke extracts.The multiple mechanisms involved in the formation of chromosomal instability and eventually evolved into malignant transformation in bronchial epithelial cells induced by coal tar pitch.