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NCI Researchers Use Mouse Tumor Cell Lines For Time-, Money-Saving Dual-Reporter Assay

Investigators at the National Cancer Institute have developed a luciferase dual-reporter assay system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell-cycle progression as evidenced by the activity of the human E2F1 promoter in vitro.
The team used murine KR158 tumor cells from a grade III NPcis aggressive anaplastic astrocytoma to generate a green and red luciferase (G/R-luc) dual-reporter system that can simultaneously assess activity of the human E2F1 promoter and cellular cytotoxicity in a single high-throughput assay.
By distinguishing cytostatic compounds from those that are cytotoxic during the initial screen, the G/R-luc assay could reduce the time and cost required to screen compound libraries, an NCI official told CBA News this week.   
“One of the advantages of this assay is that because we are using an inbred strain of mice, we can work directly with these cells in immunocompetent animals,” said Karlyne Reilly, an investigator and head of the Genetic Modifiers of Tumorigenesis section of the Mouse Cancer Genetics Program at NCI.
One of the limitations to some of the current assays is that human tumor cells must be studied in immune incompetent animals; otherwise the animals reject the tumor cells, because they recognize them as foreign.
“We can start combining different studies to look at what directly affects the tumor cells, and put the tumor cells into animals and look at how the immune system might combine with them,” said Reilly, who is also a co-author of a study describing the assay that appeared online last in the Journal of Biomolecular Screening.

“The assay saves time and money, because we are assaying for two things at the same time.”

The dual luciferase assay created by the NCI scientists is a rapid, low-background system that can be run in a high-throughput system, such as a 384-well format, said Reilly. “The assay saves time and money, because we are assaying for two things at the same time,” she added.
She said that typically, when researchers screen a drug, they want to understand its LC50 and GI50 values, and the two luciferases allow her group to assay them directly at the same time.
Most of the current methods employ human cell lines, which for the most part are glioblastomas, a kind of brain tumor, said Reilly; very few cell lines from anaplastic astrocytoma, another kind of brain tumor, are available.
In addition, most researchers use an Alamar blue assay, which measures the metabolic rate of cells, so it gives investigators an indication of how fast the cells are proliferating in terms of how fast they are using energy. “This technique we developed gives us an alternate window on that existing technique, because we are measuring the cell cycle directly,” said Reilly.
Model Mice
The NCI scientists developed a mouse model of anaplastic astrocytoma, which is relatively common in the human population, “to better understand [its] basic biology,” Reilly explained.
The researchers created the model by combining a mutation in Trp53 found in many human astrocytomas, and a mutation in the gene Nf1. Nf1 is a gene that is mutated in neurofibromatosis.
According to the investigators, mice carrying mutations in Trp53 and Nf1 on the same chromosome have been characterized as a mouse model of NF1 and astrocytoma.
The gene product of Nf1, neurofibromin, is involved in downregulating the RAS signaling pathway linking growth factor signals to cellular proliferation.
“What we have really made is a mouse model where we get upregulation of RAS signaling and loss of the p53 signaling pathway. This makes a very cancer-prone mouse,” said Reilly.  
These mice develop spontaneous astrocytomas and several other types of tumors that appear very similar to human tumors under the microscope, said Reilly.
Currently, individuals with these tumors are treated with surgery, but the tumors are diffusely infiltrated, making it difficult to completely excise the cancer.
“To take our mouse model further, we have taken cell lines from these tumors to study in culture, and what we have done in the paper is to modify those tumor cells to make them a little bit more specialized for preclinical screening of potential therapies,” said Reilly.
To make mouse tumor-derived cell lines for use in screening compounds, the NCI investigators stably transfected pHygro-Ef-CBGluc into grade III KR158 mouse astrocytoma cells to generate G-luc astrocytoma cells. They then stably transfected pPuro-CMV-CBRluc into the G-luc astrocytoma cells to create the G/R-luc dual-reporter cell line. 
The NCI researchers are currently using the G/R-luc dual-reporter cell line for high-throughput screening. “We have access to a lot of compound libraries at NCI that are very specialized, so that is what we have been focusing on,” Reilly said.
She added that she thinks it would be interesting to see if these cells can be used in vivo, because “we are taking a commonly used luminescence-based assay and filtering it, or separating it out into two different colors.” It is currently unclear if that can be done in a living animal.
However, “that would be a very powerful system, so it would be something that we would like to explore in the future a little bit,” Reilly said.

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