In the current work we are investigating how rapamycin (Rap) and sodium butyrate (NaBu) cause an increase in monoclonal antibody (MAb) production in a hybridoma cell line while delaying cell death. It has been shown that when the cell line is exposed to Rap and NaBu for 72 hrs there is an increase in MAb production. The knowledge that a compound causes an increase in MAb production is key, but it is the determination of which pathways are important in up regulating MAb production which will allow long-term goals to be obtained. For example, once the pathways are identified and understood then new cell lines may be engineered to capitalize on the ability to increase the production of MAbs.

In order to investigate the increase in MAb production caused by adding Rap and NaBu a series of questions needs to be explored. The first question that will be sought is whether or not the total protein production has been changed after exposure to the two compounds. By comparing the cell lysate of cells exposed to rapamycin and sodium butyrate to the lysate of the compound free controls through the use total protein assays we hope to determine if the total protein production has changed. We will also investigate whether exposure to rapamycin and sodium butyrate affects the secretated protein levels of the cultures. BCA assays along with ELISAs for MAb concentration will allow us to explore whether or not the secretion step in the MAb production pathway has changed.

Another question that will be addressed is whether transcription and translation are changed when an increase in MAb production is caused by rapamycin and sodium butyrate. Real time PCR will be conducted to see if the IgG mRNA levels are altered. One might theorize that if the IgG mRNA levels increase when exposed to MAb enhancing agents that transcription must be a key step in increasing the final MAb titer. The translation step of the pathway will be analyzed by using western blots to determine if the relative amounts of IgG Heavy (H) and IgG Light (L) chains are changed when a MAb enhancer is added to the culture. Note that the supernatant will also be sampled to identify any possible changes in the level of secretated IgG H and L chains.

The last thing that will be explored in this work is the effect of rapamycin and sodium butyrate on their respective main targets, the FRAP pathway and the inhibition of histone deacetylation (HDAC). Western blots will be used to determine whether a 250 ìM concentration of sodium butyrate is high enough to cause the inhibition of HDAC, which influences transcription. Rapamycin's affect on the FRAP pathway at a concentration of 100 nM will be probed using western blots to determine if the phosphorylation level of p70s6k and 4E-BP1 changes.

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