agar and Uterine-Cervical-Neoplasms

The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.

Topics: Agar; Alphapapillomavirus; Cell Transformation, Neoplastic; Female; Humans; MicroRNAs; Papillomaviridae; Papillomavirus Infections; Uterine Cervical Neoplasms

Topics: Agar; Biopsy; Cell Culture Techniques; Colony-Forming Units Assay; Female; Fibroblasts; Humans; Radiation Tolerance; Uterine Cervical Neoplasms

Radiation survival curves have been generated for 3 human tumor cell lines as a means of comparing and evaluating the validity of human tumor soft-agar clonogenic assays. The assays investigated were the Hamburger-Salmon, Courtenay-Mills, Courtenay-Mills plus additions, soft agar (no additions), and soft agar plus additions. The additions were formulated to supplement the media used in soft agar assays of primary ovarian and cervical carcinoma specimens. Supplementing the media with additions led to a 2- to 3-fold increase in PE of CaSki cells but had no effect on the PEs of ME180 and OWI cells. Radiation survival curves were similar in all assays for CaSki and OWI but differed for ME180 cells. For ME180 cells, the Courtenay-Mills and soft agar assays plus additions produced the most radioresistant curves (Do = 2.2 Gy); the cells were more responsive when assayed by the Hamburger-Salmon method (Do = 1.5 Gy), and the soft agar and Courtenay-Mills assays gave the most radiosensitive curves (Do = 1.2 Gy). These results demonstrate that the PE of human tumor cell lines may be increased with no effect on radiation survival; radiation survival may be altered without changes in PE and neither may be altered by applying modifications and supplements to existing clonogenic assays.

Topics: Adenocarcinoma; Agar; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Colony-Forming Units Assay; Female; Humans; Neoplasms; Ovarian Neoplasms; Tumor Stem Cell Assay; Uterine Cervical Neoplasms

Topics: Agar; Animals; Arboviruses; Cell Line; Cell Membrane; Cell Nucleus; Chick Embryo; Cricetinae; Culture Techniques; Cytopathogenic Effect, Viral; Cytoplasm; Female; HeLa Cells; Humans; Kidney; Mice; Microscopy, Electron; Uterine Cervical Neoplasms; Virus Replication

Topics: Agar; Carcinoma; Early Diagnosis; Female; Humans; Uterine Cervical Neoplasms