Weston E. Spencer and Dr. Daniel L. Simmons, Chemistry and Biochemistry
Among the fastest moving areas of research today is that of RNA splicing and protein synthesis. Recently, a new gene (Cyclooxygenase-2) was discovered that encodes a protein in chicken embryo fibroblasts (CEF-147) believed to be involved in the synthesis of prostaglandins. Prostaglandins are naturally occurring fatty acids that can lower blood pressure, regulate acid secretion of the stomach, regulate body temperature, control inflammation, and can affect the action of certain hormones. This novel gene is found in tumor cells in the colon. When the tumor cells become malignant the gene becomes extremely active, thus producing more of its corresponding protein which in turn increases the rate of prostaglandin synthesis. This increase in prostaglandin synthesis is what can elicit some of the symptoms associated with colorectal cancer. In order to study the effects of this process we must understand the mechanism by which this gene becomes active. To do this we must induce cells containing this gene to produce it in great quantities. This is accomplished by mitogen stimulation or by activation of a cancer gene called Src, both of which turn on cellular division at a high rate.
Chicken Embryo Fibroblasts (CEF) have a Cyclooxygenase-2 (Cox-2) gene with several introns. The introns closest to the 3′ end are spliced normally but the one closest to the 5′ end is not. This 5′ intron can be induced to splice by mitogen stimulation or specifically, by activation of pp60v-src in CEF cells infected with tsNY72-4RSV (a temperature-sensitive mutant of Rous Sarcoma Virus). When shifted to the permissive temperature of 35-370C (from 41.50C) splicing of this intron is induced and subsequent formation of CEF-147 protein is observed. CEF-147 is believed to be involved in catalyzing the rate-limiting steps in prostaglandin synthesis. The location in the cell of this splicing is of interest as well as the possibility of transfecting RNA into CEF cells as a means of generating large quantities of specific proteins. This second aspect could theoretically be used as a treatment for disease by augmenting the activity of certain key enzymes.
Three constructs were made for use in generating cRNA for transfection. One containing a fully spliced form of Cox-2 fused with the reporter gene luciferase, one containing an unspliced form of Cox-2 also fused with luciferase, and one containing only the gene for -galactosidase to act as a baseline measurement of activity. The cRNA transcripts generated from these constructs were transfected into CEF cells and the activity of the Cox-2 protein was assayed via luciferase assay. Because of the labile nature of RNA extreme caution was exercised to preserve the integrity of the generated cRNA strands. The cRNA strands were transfected into the CEF cells using a cationic liposome called LipofectinTM and put at 370C. Lipofectin is normally used as a means for transfecting DNA into cells. The small, positively charged lipofectin complexes coat the negatively charged nucleic acid and adhere to the cell membrane. Upon fusing with the cell membrane, the nucleic acid is released to the inside of the cell.
This transfection experiment was repeated several times and the cells were allowed to stay at 370C for varying amounts of time. The results varied widely probably due to differing degrees of RNA degredation in the separate experiments. No splicing seemed to occur in the cytosol 285 and the cRNA generated from the unspliced construct did not report any activity above baseline. This could be due to the fact that splicing does occur in the nucleus and the RNA was degraded in the cytosol. The cRNA generated from the spliced construct did report minimal activity above baseline in some trials but not consistently. The practicality of transfecting RNA into cells as a method of treatment for disease is limited because of the labile nature of RNA.
The determination of the location of the splicing in the cell still needs to be done. It is hypothesized that some splicing functionality is present in the cytosol. To determine this, unspliced cRNA trancripts must be transfected into the cells and after some time the RNA can be processed in cytosolic and nuclear fractions. These RNA samples can be run on a denaturing agarose gel and subsequently blotted onto a membrane where the samples can be probed with a radioactively labeled, fully-spliced construct that will hybridize with any fully-spliced RNA.1 The resulting bands can then be quantitatively evaluated and a determination can be made as to where the splicing occurs. If it is found that the RNA can be spliced in the cytosol then the prospect of transfecting RNA into the cell becomes more real and further experimentation can be done in this regard.
The reliability of RNA transfection as a treatment for disease is still in question and it may or may not prove to be a beneficial way of treating conditions such as colorectal cancer. The continuing research over the next couple of years will determine whether RNA or other genetic material can be used as a widespread means of combating such cellular related diseases.
References
- Xie, W., Chipman, J. G., Roberston, D. L., Erikson, R. L. & Simmons, D. L. (1991) Proc. Natl. Acad. Sci. USA 88, 2692-2696.