Microinjection strategies for the study of mitogenic signaling in mammalian cells.

Abstract

First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture. In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner. A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts. These strategies include: 1. Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain. 2. Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities. 3. Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules. In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response. The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4). For in vivo studies, the c-fos promoter offers several unique advantages. Primarily, it is easy to manipulate. In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours. Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5). Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress. In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression. However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation). As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off.