<i>REV3L</i>, a Promising Target in Regulating the Chemosensitivity of Cervical Cancer Cells

<div><p>REV3L, the catalytic subunit of DNA Polymerase ζ (Polζ), plays a significant role in the DNA damage tolerance mechanism of translesion synthesis (TLS). The role of <i>REV3L</i> in chemosensitivity of cervical cancer needs exploration. In the present study, we evaluated the expression of the Polζ protein in paraffin-embedded tissues using immunohistochemistry and found that the expression of Polζ in cervical cancer tissues was higher than that in normal tissues. We then established some cervical cancer cell lines with <i>REV3L</i> suppression or overexpression. Depletion of <i>REV3L</i> suppresses cell proliferation and colony formation of cervical cancer cells through G₁ arrest, and <i>REV3L</i> promotes cell proliferation and colony formation of cervical cancer cells by promoting G₁ phase to S phase transition. The suppression of <i>REV3L</i> expression enhanced the sensitivity of cervical cancer cells to cisplatin, and the overexpression of <i>REV3L</i> conferred resistance to cisplatin as evidenced by the alteration of apoptosis rates, and significantly expression level changes of anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia sequence 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl) and proapoptotic Bcl-2-associated x protein (Bax). Our data suggest that <i>REV3L</i> plays an important role in regulating cervical cancer cellular response to cisplatin, and thus targeting <i>REV3L</i> may be a promising way to alter chemosensitivity in cervical cancer patients.</p></div

<i>REV3L</i> knockdown sensitizes cervical cancer cells to cisplatin.

<p>(A) Suppression of <i>REV3L</i> confered cervical cancer cells more sensitive to the cytotoxic effect of cisplatin. (B) Suppression of <i>REV3L</i> showed hypersensitivity to cisplatin. Cells were treated with the indicated concentrations of cisplatin for 24 h, followed by staining with Annexin V–fluorescein isothiocyanate (FITC) and propidiumiodide (PI) for early apoptotic cells (Annexin V+ PI–). Early apoptotic rates of the <i>REV3L</i>-suppression cells were significantly higher than those of the vector control cells under the same condition. (C) The percentage of apoptotic cells induced by cisplatin. Data are means of three independent experiments ± SEM. * <i>P</i>< 0.05, **<i>P</i>< 0.01.</p

<i>REV3L</i> overexpression rendered resistance to cisplatin in cervical cancer cells.

<p>(A) Overexpression of <i>REV3L</i> rendered cervical cancer cells resistant to cisplatin. (B) Detection of apoptotic cells by flow cytometry. Overexpression of <i>REV3L</i> inhibited apoptosis induced by cisplatin. (C) The percentage of apoptotic cells induced by cisplatin. Data are means of three independent experiments ± SEM. * <i>P</i>< 0.05.</p

Polζ expression in cervical cancer specimens and normal cervical tissues and mRNA expression of <i>REV3L</i> in cervical cancer cell lines.

<p>(A) Polζ-positive (upper, left panel) and Polζ-negative (upper, right panel) expression in cervical cancer specimens, Polζ-positive (lower, left panel) and Polζ-negative (lower, right panel) expression in normal cervical tissues. (B) The clinical characteristics and the status of Polζ expression of cervical cancer patients and patients with nomal cervix. The mean Polζ expression was higher in the cervical cancer tissues than that in the normal cervical tissues. (C)Real time PCR analysis for <i>REV3L</i> mRNA expression in four cervical cancer cell lines. <i>REV3L</i> expression was higher in HeLa and SiHa cells than in MS751 and ME180 cells. (D) Reverse transcription PCR analysis for <i>REV3L</i> mRNA expression in short hairpin RNA transfected negative control cells (shGFP), shREV3L cells, <i>REV3L</i>-overexpressing cells, and vector control cells. <i>REV3L</i> expression was significantly decreased in shREV3L cells and was increased in the <i>REV3L</i>-overexpressing cells compared with control cells.</p

Influence of upregulation or downregulation of <i>REV3L</i> on cell proliferation, cell cycle, and colony formation ability.

<p>(A) Depletion of <i>REV3L</i> suppressed cell proliferation of HeLa shREV3L cells and SiHa shREV3L cells. (B) Enhancement of <i>REV3L</i> promoted cell proliferation of MS751 <i>REV3L</i> and ME180 <i>REV3L</i> cells. (C) Depletion of <i>REV3L</i> induced G₁ arrest in HeLa shREV3L cells. (D) Enhancement of <i>REV3L</i> promoted G₁ phase to S phase transition in ME180 <i>REV3L</i> cells. (E) Depletion of <i>REV3L</i> suppressed colony formation of HeLa shREV3L and SiHa shREV3L cells. (F) Enhancement of <i>REV3L</i> promoted colony formation of MS751 <i>REV3L</i> and ME180 <i>REV3L</i> cells. Data are means of three independent experiments ± SEM. * <i>P</i>< 0.05.</p

Different inhibitory concentrations (IC) of cisplatin in HeLa, SiHa shREV3L cells and control cells.

<p>IC30, 30% inhibitory concentration of cisplatin; IC50, 50% inhibitory concentration of cisplatin; IC70, 70% inhibitory concentration of cisplatin.</p><p>*<i>P<</i>0.05.</p><p>Different inhibitory concentrations (IC) of cisplatin in HeLa, SiHa shREV3L cells and control cells.</p