Development of a fluorescence-based cellular apoptosis reporter.

Evasion of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer agents is the induction of cell death. With the heterogeneity of cancer becoming increasingly apparent, to understand drug mechanisms of action and identify combination therapies in cell populations, the development of tools to assess drug effects at the single cell level is a necessity for future preclinical drug development. Herein we describe the development of pCasFSwitch, a genetically encoded reporter construct designed to identify cells undergoing caspase-3 mediated apoptosis, by a translocation of a GFP signal from the cell membrane into the nucleus. Anticipated cellular distribution was demonstrated by use of confocal microscopy and cleavage by caspase-3 was shown to be required for the translocation of the GFP signal seen in apoptotic cells. Quantification of apoptosis using the construct revealed similar levels to that obtained with a commercially available apoptosis imaging agent (22.6% versus 20.3%). Moreover, we demonstrated its capacity for use in a high-throughput setting making it a powerful tool for drug development pipelines.

Diversity of matriptase expression level and function in breast cancer.

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.

Two-color photoactivatable probe for selective tracking of proteins and cells.

We report the development and application of photoactivatable Green Cherry (G(PA)C), the first genetically encoded "continuously red-photoactivatable green" two-color probe for live cell imaging. G(PA)C is unique in that it enables real-time tracking of selected subpopulations of proteins and organelles in the cell or of cells within tissues and whole organisms, with constant reference to the entire population of the probe. Using G(PA)C-zyxin as proof of utility, we obtained new insights into the dynamic movement of the cytoskeletal protein zyxin. We show that zyxin is continuously and rapidly recruited from the cytosol into established focal adhesions. It can also move rapidly within a given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the dynamic nature of proteins within these structures. The in vivo utility of G(PA)C is exemplified by tracking hemocyte movements using a versatile transgenic Drosophila model engineered to express G(PA)C in tissues and cells of interest under the control of the GAL4-inducible promoter.

Blocking phosphoinositide 3-kinase activity in colorectal cancer cells reduces proliferation but does not increase apoptosis alone or in combination with cytotoxic drugs.

In response to growth factors, class IA phosphoinositide 3-kinases (PI3K) phosphorylate phosphatidylinositol-4,5-bisphosphate, converting it to phosphatidylinositol-3,4,5-trisphosphate to activate protein kinase B/Akt. This is widely reported to promote tumorigenesis via increased cell survival, proliferation, migration, and invasion, and many tumor types, including colorectal cancer, exhibit increased PI3K signaling. To investigate the effect of inhibiting PI3K and as an alternative to the use of small molecular inhibitors of PI3K with varying degrees of selectivity, HT29 and HCT116 colorectal cancer cells bearing mutant PIK3CA were generated that could be induced with doxycycline to express synchronously a dominant negative subunit of PI3K, Deltap85alpha. On induction, decreased levels of phosphorylated protein kinase B were detected, confirming PI3K signaling impairment. Induction of Deltap85alpha in vitro reduced cell number via accumulation in G(0)-G(1) phase of the cell cycle in the absence of increased apoptosis. These effects were recapitulated in vivo. HT29 cells expressing Deltap85alpha and grown as tumor xenografts had a significantly slower growth rate on administration of doxycycline with reduced Ki67 staining without increased levels of apoptotic tissue biomarkers. Furthermore, in vitro Deltap85alpha expression did not sensitize HT29 cells to oxaliplatin- or etoposide-induced apoptosis, irrespective of drug treatment schedule. Further analysis comparing isogenic HCT116 cells with and without mutation in PIK3CA showed no effect of the mutation in either proliferative or apoptotic response to PI3K inhibition. These data show in colorectal cancer cells that PI3K inhibition does not provoke apoptosis per se nor enhance oxaliplatin- or etoposide-induced cell death.

Difluoro analogue of UCS15A triggers activation of exogenously expressed c-Src in HCT 116 human colorectal carcinoma cells.

UCS 15A, an antibiotic produced by Streptomyces sp., has been reported to specifically disrupt SH3 domain-mediated interactions in eukaryotic cells. Interestingly, in the case of the non-receptor tyrosine kinase Src, UCS15A was effective in suppressing the SH3 domain-mediated intermolecular rather than intramolecular interactions, and thus prevented Src interactions with certain downstream effectors without affecting Src kinase activity. Here the synthesis of a novel difluoro analogue of UCS15A is described. The effects of this compound (8) on Src activity were tested in HCT 116 colorectal carcinoma cells engineered for inducible expression of c-Src. The presence of compound (8) resulted in the increased activity of the induced c-Src implicating that (8) acts as a c-Src activator in vivo. These observations are supported by computer modelling studies which suggest that the aldehyde group of (8) may covalently bind to a lysine residue in the SH2-kinase linker region situated in the proximity of the SH3 domain, which could promote a conformational change resulting in increased Src activity.

Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells.

Phosphorylation plays an important role in regulation of protein kinase C delta (PKCdelta). To date, three Ser/Thr residues (Thr 505, Ser 643, and Ser 662) and nine tyrosine residues (Tyr 52, Tyr 64, Tyr 155, Tyr 187, Tyr 311, Tyr 332, Tyr 512, Tyr 523, and Tyr 565) have been defined as regulatory phosphorylation sites for this protein (rat PKCdelta numbering). We combined doxycycline-regulated inducible gene expression technology with a hypothesis-driven mass spectrometry approach to study PKCdelta phosphorylation pattern in colorectal cancer cells. We report identification of five novel Ser/Thr phosphorylation sites: Thr 50, Thr 141, Ser 304, Thr 451, and Ser 506 (human PKCdelta numbering) following overexpression of PKCdelta in HCT116 human colon carcinoma cells grown in standard tissue culture conditions. Identification of potential novel phosphorylation sites will affect further functional studies of this protein, and may introduce additional complexity to PKCdelta signaling.

Increases in c-Yes expression level and activity promote motility but not proliferation of human colorectal carcinoma cells.

Increases in the levels and/or activity of nonreceptor tyrosine kinases c-Src and c-Yes are often associated with colorectal carcinogenesis. The physiological consequences of increased c-Yes activity during the early and late stages of tumorigenesis, in addition to the degree of redundancy between c-Yes and c-Src in colorectal cancer cells, remain elusive. To study the consequences of increases in c-Yes levels and activity in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Yes levels and activity can be inducibly increased by a tightly controlled expression of wild-type c-Yes or by constitutively active mutants of c-Yes, c-YesY537F, and c-Yes Delta t6aa. c-Yes induction resulted in increased cell motility but did not promote proliferation either in vitro or in vivo. These results suggest that in later stages of colorectal carcinogenesis, elevations in c-Yes levels/activity may promote cancer spread and metastasis rather than tumor growth.

Comparison of doxycycline delivery methods for Tet-inducible gene expression in a subcutaneous xenograft model.

Doxycycline (Dox) controlled Tet systems provide a powerful and commonly used method for functional studies on the consequences of gene overexpression/downregulation. However, whereas Dox delivery in tissue culture in vitro is relatively simple, the situation in vivo is more complex. Several methods of Dox delivery in vivo have been described-e.g., in drinking water containing alcohol, in drinking water containing various concentrations of sucrose, and in feed. Unfortunately there are no reports directly comparing the advantages and disadvantages of these diverse methods, and there is no generally accepted standard. We therefore compared four non-invasive methods of Dox delivery in vivo-in drinking water, by gavage, as a jelly, and in standard feed. To assess the delivery of Dox by these methods, we used a subcutaneous xenograft model based on colorectal carcinoma cells engineered for Dox-inducible expression of an activated mutant of c-Src and the luciferase reporter gene. Our results indicate that feed represents the most favorable method of Dox administration.

Tetracycline regulated systems in functional oncogenomics.

The increasing number of proteomic and DNA-microarray studies is continually providing a steady acquisition of data on the molecular abnormalities associated with human tumors. Rapid translation of this accumulating biological information into better diagnostics and more effective cancer therapeutics in the clinic depends on the use of robust function-testing strategies. Such strategies should allow identification of molecular lesions that are essential for the maintenance of the transformed phenotype and enable validation of potential drug-targets. The tetracycline regulated gene expression/ suppression systems (Tet-systems) developed and optimized by bioengineers over recent years seem to be very well suited for the function-testing purposes in cancer research. We review the history and latest improvements in Tet-technology in the context of functional oncogenomics.

Increases in c-Src expression level and activity do not promote the growth of human colorectal carcinoma cells in vitro and in vivo.

The levels and activity of c-Src in colorectal cancer cells increase steadily during the course of colorectal carcinogenesis and are most highly elevated in advanced metastatic disease. However, the effects of increases in c-Src activity on the proliferation of colorectal cancer cells during early and late stages of tumorigenesis remain elusive. To study the consequences of increases in c-Src levels and activity on the growth of colorectal cancer cells in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Src levels and activity could be inducibly increased by a tightly controlled expression of wild-type c-Src or of the constitutively active mutant of c-Src, c-SrcY527F. Src induction activated multiple signaling pathways (often associated with a proliferative response) but promoted neither cell proliferation in vitro nor tumor growth in a xenograft model in vivo. These results indicate that, in more advanced stages of colorectal carcinogenesis, increases in c-Src levels and activity are likely to have functions other than the direct promotion of tumor growth.

Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2.

Tet-on cell lines engineered to stably express doxycycline (Dox)-regulated reverse transcriptional transactivator (rtTA) have many applications in biomedical research and biotechnology. Unfortunately, construction and maintenance of such cells often proves to be costly, labor intensive and ineffective. Moreover, the Tet-on clones generated using standard methodology were often unstable and frequently displayed significantly changed physiological properties compared with their parental cells. Here we describe an optimized protocol for generation of Tet-on cells. The protocol is based on the use of a recently developed pN1p beta actin-rtTA2S-M2-IRES-EGFP vector (where IRES is an internal ribosome entry site) and permits relatively inexpensive construction of many Tet-on clones with essentially 100% efficiency. The method is well suited for 'difficult' cell lines displaying genetic instability and high levels of epigenetic silencing. The constructed Tet-on cells remain stable with time in the absence of any selection agents, are easy to monitor and preserve the characteristics of parental cells. The protocol can be completed in 2 months.

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression.

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.