Discussion

We describe a rapid and reliable procedure for quantitation of intracellular proteins modulated by targeted therapy. The approach presents several advantages over conventional Western blot analysis. Costaining specific cell populations with surface markers allows monitoring of total and phosphory-lated intracellular protein levels for each subpopulation. Quantitative multiparameter information is generated for each sample per analyte for every 100 cells. Technically, this approach requires much less sample volume and minimal manipulation of sample, maintaining the integrity of the proteins of interest.

The modulation of intracellular proteins can be manifested as changes in cellular positivity or fluorescence intensity. Combining the two factors into an index value provides a unit for measuring changes in total and phosphorylation levels of proteins. The ratio of phosphorylated-to-total protein changes could provide insight on drug efficacy.

The same approach can be used to monitor intracellular protein modulation other than phosphorylation. For example, histone acetylation can be quantitated in patients undergoing treatment with histone deacetylase (HDAC) inhibitors. Monitoring levels of these downstream factors may provide a means of customizing therapy by predicting which patients will respond to a given treatment and determining optimal dosage.

4. Notes

1. A novel bead-based approach to the direct and quantitative measurement of fusion proteins such as BCR-ABL is described in Chapter 12.

2. When available, rabbit and mouse isotype controls are used. Nonstaining cell populations within the sample can be used as negative controls.

3. Several other fixation and permeabilization reagents also available.

4. Though it is preferable to acquire 5000 events, depending on the sample, a smaller number may provide adequate quantitative information.

5. The index is derived by multiplying the percentage positivity by the ABC. This takes into account both changes.

Fig. 3. Histograms representing the effect of an imatinib mesylate on drug insensitive cell lines (A), a drug-sensitive BCR-ABL positive cell line (B) and CD34+ blast cells from a chronic myelogenous leukemia patient (C). Cells were fixed and stained with phospho-CRKL (0.05 mg/50 p,L). The grey (pretherapy) and black (posttherapy) lines denote immunofluorescence before and after treatment, respectively. Index values of cells are noted pre- and posttherapy.

Fig. 3. Histograms representing the effect of an imatinib mesylate on drug insensitive cell lines (A), a drug-sensitive BCR-ABL positive cell line (B) and CD34+ blast cells from a chronic myelogenous leukemia patient (C). Cells were fixed and stained with phospho-CRKL (0.05 mg/50 p,L). The grey (pretherapy) and black (posttherapy) lines denote immunofluorescence before and after treatment, respectively. Index values of cells are noted pre- and posttherapy.

References

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