Vučković Nika, Hoppe-Seyler Karin, Riemer Angelika B
Immunotherapy and Immunoprevention, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
Molecular Vaccine Design, German Center for Infection Research (DZIF), Partner Site Heidelberg, 69120 Heidelberg, Germany.
Cancers (Basel). 2023 Jul 27;15(15):3810. doi: 10.3390/cancers15153810.
Cervical cancer is the fourth leading cause of cancer deaths in women, with over 340,000 women dying from this disease in 2020. Almost all cases have an underlying persistent infection with an oncogenic high-risk type of human papillomavirus (HPV), mainly HPV16. While cervical squamous cell carcinoma is hardly ever HPV-negative, a small subset of adenocarcinoma exhibits absence of HPV, even after disproval of false-negative testing results due to low viral load. This proportion is evident in many cervical cancer studies and is reflected in the repertoire of model cell lines commonly used in research. As the viral origin of cervical cancer makes it a disease preventable and potentially treatable by immunotherapeutic approaches, it is the focus of many studies. For pertinent research, both a broad set of HPV-infected cervical carcinoma models are required, as well as stringent negative controls. A ubiquitously used HPV-negative cervical adenocarcinoma cell line is C-33A. Another cervical cancer cell line is available for purchase from the American Type Culture Collection (ATCC), namely DoTc2 4510, described to be HPV-negative and thus as a model for a rare gynecological malignancy. Here, we present findings proving that DoTc2 4510 is, in fact, an HPV16-positive cell line. This we assessed using a highly sensitive nested multiplex PCR protocol adapted for the identification of 12 carcinogenic HPV types and a second PCR targeting the HPV16 oncogenes E6 and E7. Subsequently, the protein expression of E6 and E7 was examined, as well as the expression of their target proteins p53, p21, and p16, to assess E6/E7 functionality. Finally, to attest to the survival dependence of DoTc2 4510 cells on HPV16, we performed an HPV16 E6/E7-targeted siRNA knock-down, which indeed led to senescence induction. Together, these findings demonstrate that DoTc2 4510 is an HPV16-transformed cell line.
宫颈癌是女性癌症死亡的第四大主要原因，2020年有超过34万名女性死于这种疾病。几乎所有病例都存在致癌性高危型人乳头瘤病毒（HPV）的持续性感染，主要是HPV16。虽然宫颈鳞状细胞癌几乎从不为HPV阴性，但一小部分腺癌即使在因病毒载量低而排除假阴性检测结果后仍表现为HPV阴性。这一比例在许多宫颈癌研究中都很明显，并反映在研究中常用的模型细胞系中。由于宫颈癌的病毒起源使其成为一种可通过免疫治疗方法预防和潜在治疗的疾病，因此它是许多研究的重点。对于相关研究，既需要广泛的HPV感染的宫颈癌模型，也需要严格的阴性对照。一种广泛使用的HPV阴性宫颈腺癌细胞系是C-33A。另一种宫颈癌细胞系可从美国典型培养物保藏中心（ATCC）购买，即DoTc2 4510，据称是HPV阴性，因此作为一种罕见妇科恶性肿瘤的模型。在此，我们展示的研究结果证明，DoTc2 4510实际上是一种HPV16阳性细胞系。我们使用一种经过高度敏感的巢式多重PCR方案进行评估，该方案适用于鉴定12种致癌性HPV类型，并使用针对HPV16癌基因E6和E7的第二种PCR。随后，检测E6和E7的蛋白表达以及它们的靶蛋白p53、p21和p16的表达，以评估E6/E7的功能。最后，为了证明DoTc2 4510细胞对HPV16的生存依赖性，我们进行了针对HPV16 E6/E7的siRNA敲低，这确实导致了衰老诱导。总之，这些研究结果表明DoTc2 4510是一种HPV16转化的细胞系。
