Role of Epithelial-Mesenchymal Transition in Tumor Progression.

Epithelial-mesenchymal transition (EMT) is a fundamental process of morphogenesis whereby epithelial cells acquire the mesenchymal phenotype. Multiple data suggest a critical role of EMT in tumor progression. In carcinomas, EMT can be initiated and promoted by many oncogenic signaling pathways, hypoxia, and signals of tumor microenvironment resulting in epithelial cells losing their cell polarity and cell-cell adhesion and gaining the migratory and invasive properties. Downregulation of expression of the cell adhesion protein E-cadherin is considered a poor prognostic factor in cancer. Many tumors are characterized by incomplete EMT, where tumor cells acquire mesenchymal characteristics but retain their epithelial markers, in particular, E-cadherin. In cells with the hybrid epithelial-mesenchymal phenotype, E-cadherin is accumulated in adherens junctions which are less stable than adherens junctions in normal epithelial cells. E-cadherin-based adherens junctions are essential for efficient collective migration and invasion of carcinoma cells, and their survival in metastasis. The plasticity of the hybrid epithelial-mesenchymal phenotype improves adaptive capabilities of cancer cells. By undergoing EMT, carcinoma cells become resistant to chemotherapy and acquire the ability to suppress immune response. Emergence of cancer stem cells after EMT activation has been observed in many types of carcinoma.

Disruption of actin microfilaments by cytochalasin D leads to activation of p53.

Activation of p53 plays a central role in the cell's response to various stress signals. We investigated whether p53 is activated upon disruption of actin microfilaments, caused by cytochalasin D (CD). We show that treatment with CD leads to accumulation of p53 in the cells and activation of p53-dependent transcription. Treatment with CD led to arrest of G1-to-S transition in cells retaining wild-type p53, while cells with inactivated p53 showed partial rescue from it. CD also induces apoptosis in p53+/+, but not in p53-/- cells. The obtained data suggest that disruption of the actin microfilaments activates p53-dependent pathways.

Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation.

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.