Involvement of DNA polymerase β overexpression in the malignant transformation induced by benzo[a]pyrene

Abstract

Objective: To explore the relationship between DNA polymerase β (pol β) overexpression and benzo[a]pyrene (BaP) carcinogenesis.

Methods: Firstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase β (pol β cell) and high level of pol β (pol β oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol β. Secondly, malignant transformation of pol β cells by low concentration of BaP (20 μM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol β cells (named pol β-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay.

Results: Pol β cells were successfully transformed into malignant pol β-T cells by an exposure to low concentration of BaP for 6 months. Pol β-T cells exhibited increased levels of pol β gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol β oe cells.

Conclusion: Pol β overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis.

Figure 1. PCR products of pol β and pol β oe cells amplified with random primers

Random Primer P1 and Random Primer P7: M, 50 bp DNA ladder marker; Lane 1 and 2, pol β cells exposed to 0 (0.1% v/v DMSO) and 20 μM BaP, respectively; Lane 3 and 4, pol β cells exposed to 0 (0.1% v/v DMSO) and 20 μM BaP, respectively; Lane 5 and 6, pol β oe cells exposed to 0 (0.1% v/v DMSO) and 20.00 μM BaP, respectively. Random Primer P2 and Random Primer P5: M, 50 bp DNA ladder marker; Lane 1 and 2, pol β cells exposed to 0 (0.1% v/v DMSO) and 20.00 μM BaP, respectively; Lane 3 and 4, pol β oe cells exposed to 0 (0.1% v/v DMSO) and 20 μM BaP, respectively; Lane 5 and 6, pol β oe cells exposed to 0 (0.1% v/v DMSO) and 20.00 μM BaP, respectively.

Figure 2. Morphological alterations of cells grown in soft agar (100×)

Control cells: 0 μM BaP (0.1% v/v DMSO) treated pol β cells served as solvent control; Transformed cells: pol β cells treated with long time and low concentration BaP and S9 mixture coexposure.

Figure 3. Pol β mRNA level in pol β cells and pol β-T cells

M: 100 bp DNA ladder marker; Lane1: pol β cells; Lane2: pol β-T cells. Quantification of the bands intensity of PCR products was analyzed by Image J. Integral optical density value (IOV) = average optical density value of bands × area of bands. Relative optical density value (rIOV) = IOV_{pol β}/IOV_G3PDH.

Figure 4. Pol β protein level in pol β cells and pol β-T cells

Quantitative analysis was performed by Image J. Integral grayscale value (IGV) = average grayscale value of protein bands × area of protein bands. Relative integral grayscale value (rIGV) = IGV_{pol β}/IGV_β-actin.

Figure 5

Morphology of mutant colony of pol β cells and pol β-T cells (100×)

Figure 6. PCR products amplified with random primers P1, P2, P4, P5 and P6 in pol β cells and pol β-T cells

M: 50 bp DNA ladder marker; Lane 1: pol β cells; Lane 2: pol β-T cells.