DataSheet_1_Stable CDK12 Knock-Out Ovarian Cancer Cells Do Not Show Increased Sensitivity to Cisplatin and PARP Inhibitor Treatment.pdf

Cyclin-dependent kinase 12 (CDK12) is a serine/threonine kinase involved in the regulation of RNA polymerase II and in the transcription of a subset of genes involved in the DNA damage response. CDK12 is one of the most mutated genes in ovarian carcinoma. These mutations result in loss-of-function and can predict the responses to PARP1/2 inhibitor and platinum. To investigate the role of CDK12 in ovarian cancer, CRISPR/Cas9 technology was used to generate a stable CDK12 knockout (KO) clone in A2780 ovarian carcinoma cells. This is the first report on a CDK12 null cell line. The clone had slower cell growth and was less clonogenic than parental cells. These data were confirmed in vivo, where CDK12 KO transplanted cells had a much longer time lag and slightly slower growth rate than CDK12-expressing cells. The slower growth was associated with a higher basal level of apoptosis, but there were no differences in the basal level of autophagy and senescence. While cell cycle distribution was similar in parental and knockout cells, there was a doubling in DNA content, with an almost double modal number of chromosomes in the CDK12 KO clone which, however did not display any increase in γH2AX, a marker of DNA damage. We found partial down-regulation of the expression of DNA repair genes at the mRNA level and, among the down-regulated genes, an enrichment in the G2/M checkpoint genes. Although the biological features of CDK12 KO cells are compatible with the function of CDK12, contrary to some reports, we could not find any difference in the sensitivity to cisplatin and olaparib between wild-type and CDK12 KO cells.

Cyclin-dependent kinase 12（CDK12），作为一种丝氨酸/苏氨酸激酶，参与RNA聚合酶II的调控以及参与DNA损伤反应的基因子集的转录。CDK12是卵巢癌中突变频率最高的基因之一。这些突变导致功能丧失，并能预测对PARP1/2抑制剂和铂类的反应。为了研究CDK12在卵巢癌中的作用，采用了CRISPR/Cas9技术，在A2780卵巢癌细胞系中生成了一种稳定的CDK12敲除（KO）克隆。这是关于CDK12缺失细胞系的首次报道。该克隆的细胞生长速度较亲本细胞慢，克隆形成能力也较低。这些数据在体内也得到了证实，CDK12 KO移植细胞的滞后时间比CDK12表达细胞长得多，生长速率略慢。较慢的生长速度与较高的凋亡基础水平相关，但在自噬和衰老的基础水平上没有差异。尽管亲本细胞和敲除细胞在细胞周期分布上相似，但CDK12 KO克隆的DNA含量翻倍，染色体数量几乎呈双峰分布，然而，这并没有导致γH2AX（DNA损伤的标志物）的增加。我们发现DNA修复基因在mRNA水平的表达部分下调，而在下调的基因中，G2/M检查点基因富集。尽管CDK12 KO细胞的生物学特征与CDK12的功能相符合，但与一些报道相反，我们未在野生型和CDK12 KO细胞之间发现对顺铂和奥拉帕利的敏感性差异。