R Gilchrist

Site-Directed Perturbation of Protein Kinase C- Integrin Interaction Blocks Carcinoma Cell Chemotaxis

ABSTRACT Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase Cα (PKCα) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKCα V3 hinge domain. This motif is also required for a direct association between PKCα and β1 integrin. Efficient binding of β1 integrin to PKCα requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of β1 integrin was shown to block both PKCα-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKCα and β1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.

Cell cycle profiles and expressions of p21CIP1 AND P27KIP1 during myocyte development.

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

Reduction of TSG101 protein has a negative impact on tumor cell growth.

TSG101 was defined originally as a tumor‐suppressor gene, raising the expectation that absence of the encoded protein should lead to increased tumor cell growth and, perhaps, increased tumor cell aggressiveness. We have used the RNA interference (RNAi) technique to downregulate TSG101 in PC3 (prostate cancer) and MDA‐MB‐231 (breast cancer) cells. An approximately 85% selective downregulation at the protein level was achieved in both cell lines over a period of 12 days as detected by Western blotting. This treatment resulted in inhibition of tumor cell growth, with a decreased level of TSG101 causing partial cell cycle arrest at the G1/S boundary and a reduction in the rate at which cells passed from G2 through mitosis and back into G1. In both cell lines, the percentage of cells in S‐phase was reduced significantly at day 4 after the TSG101 siRNA transfection (27% vs. 41% in MDA‐MB‐231 cells; 22% vs. 39% in PC3 cells). Additionally, RNAi‐mediated downregulation of TSG101 reduced the colony formation capacities of both cancer cell lines. Rather more surprisingly, TSG101 downregulation affected the migratory activity of the MDA‐MB‐231 cells, independent of any effect on proliferation. Thus, in a Transwell assay, after 4‐hr incubation, 36.0% of control MDA‐MB‐231 cells had migrated to the lower chamber vs. 7.3% of TSG101‐downregulated cells (p < 0.001; scrambled control, 36.5%). These results show that the TSG101 gene does not comply with the usual characteristics of a tumor‐suppressor gene; rather, its expression may be necessary for activities associated with aspects of tumor progression.

The value of rapid functional assays of germline p53 status in LFS and LFL families.

We have tested two rapid assays of p53 function, namely the apoptotic assay and the FASAY as means of detecting germline p53 mutations in members of Li–Fraumeni and Li–Fraumeni-like families. Results of the functional assays have been compared with direct sequencing of all 11 exons of the p53 gene. The results show good agreement between the two functional assays and between them and sequencing. No false-positives or negatives were seen with either functional assay although the apoptotic assay gave one borderline result for an individual without a mutation. As an initial screen the apoptotic assay is not only rapid but inexpensive and very simple to perform. It would be expected to detect any germline defect that leads to loss of p53 function. The apoptotic assay could be ideal as a means of prescreening large numbers of samples and identifying those that require further investigation. The FASAY detects mutations in exons 4–10, is rapid and distinguishes between functionally important and silent mutations.