Question

It is known that some tumors are associated with tumor viruses. It remains unknown, however, whether or not other tumors such as those induced by chemical carcinogens are also associated with tumor viruses. Design a series of experiments to test the hypothesis that a chemical carcinogen-induced tumor cells contain altered genome (as compared to its unaltered state in the normal cell counterparts) and this genomic DNA alteration is responsible for the transformed phenotypes of the tumor cells. Include in your experimental design the following:

1) the overall strategy,
2) the reagents chosen including controls and why they are chosen,
3) at least four types of transformation assays, including both in vitro and in vivo assays, to be used to measure the properties of transformed cells, and
4) the strategy to interpret the results and to draw a conclusion.

Answer 1

Experimental procedure:

1- To expose cell lines to both chemical carcinogens and viruses known to produce cancer (e.g., Epstein-Barr virus, hepatitis B virus, human papillomavirus, herpesvirus-8, etc) simultaneously.

2- By using real-time PCR (RT-PCR), to amplify mRNAs for specific target oncogenes both in normal cells and in cells that acquired cancer traits such as, for example, cell proliferation and survival. During this experimental procedure, it is required to include in vitro transformational assays including 1-anchorage independence, 2-morphological transformation (where the cells are scored for lack of contact inhibition), 3-focus formation and 4-immortalization.

3- Also by RT-PCR, to amplify mRNAs for specific viral mRNAs (i.e., to determine the viral load) in cancer-related cell lines.

4- To determine if there is a statistical significance between specific oncogene mRNAs versus viral expression in cancer-related cell lines

Experimental controls:

- Negative control: normal cells (i.e., cells non-exposed to chemical carcinogens and viruses)

- Positive control: immortalized cell lines where it has been found that the target oncogenes are expressed. It is possible to obtain this genetic data from other studies (i.e., by searching this information in the corresponding scientific publications)

Step-by-step explanation:

By using this experimental procedure, it is possible to identify if there exists a correlation between the presence of viral mRNA and oncogenes previously recognized for being amplified in different types of cancers in cell lines that were capable of developing cancer traits.