Cell proliferation and oncogene expression after bile duct ligation in the rat: Evidence of a specific growth effect on bile duct cells

The proliferative response of the rat liver was measured after temporary or permanent total biliary obstruction (BDO) and in different regions after selective ligation of the lobar ducts draining the right 60% of the hepatic mass. The results were compared with those after 70% partial hepatectomy (PH). Cell proliferation was assessed globally by measuring DNA synthesis and stratified to the separate cell populations with cytostaining techniques that allowed distinction of hepatocytes, duct cells, and nonparenchymal cells (NPCs). In selected experimental groups, gene expression was determined of transforming growth factor-β1 (TGFβ-1), prothrombin, c-erb-B2, transforming growth factor alpha (TGFα), human Cyclophilin (CyP), and 28S ribosomal RNA. The stimulation of a proliferative response to total BDO required obstruction for longer than 24 hours, but after this deligation did not switch off regeneration. In the first week after permanent BDO, there was progressive infiltration of NPCs, fibrous linkage of some portal areas, and a crescendo of DNA synthesis that was obvious at 24 hours, maximal at 48 hours, and back nearly to baseline at 6 days. At the 2-day mark, the bile duct cells had a 17-fold increase in proliferation, accompanied by a threefold to fourfold increase in hepatocyte renewal. Little or no increase in expression of TGFα or the hepatocyte-specific prothrombin gene was detectable in the first 48 hours, whereas levels of the oncogene c-erb-B2 that is associated with cholangiocarcinoma were expressed from 48 to 96 hours. Livers subjected to regional BDO with or without immunosuppressive treatment with FK 506 and cyclosporine had an inflammatory reaction only on the side with ligated ducts. DNA synthesis increased in both the obstructed and freely draining lobes to approximately half the level that occurred after total BDO. The proliferation of the obstructed side was similar to the mixed duct cell/hepatocyte response after total BDO, but this almost exclusively involved duct cells on the freely draining side. In contrast to the findings after BDO, livers after PH regenerated maximally at 24 hours rather than 48 hours, had a predominantly noninflammatory hepatocyte as opposed to duct cell response, and had marked expression of the prothrombin and TGFα genes but only weakly and late of c-erb-B2 messenger RNA. The results show that the liver responds as a whole and in a biologically intelligent way to the nature of the injury inflicted on any part of it. It further implies the presence of humoral communications and control networks that assure organ homeostasis and relate this to total body homeostasis. © 1995

Differential Matrix Rigidity Response in Breast Cancer Cell Lines Correlates with the Tissue Tropism

Metastasis to a variety of distant organs, such as lung, brain, bone, and liver, is a leading cause of mortality in the breast cancer patients. The tissue tropism of breast cancer metastasis has been recognized and studied extensively, but the cellular processes underlying this phenomenon, remain elusive. Modern technologies have enabled the discovery of a number of the genetic factors determining tissue tropism of malignant cells. However, the effect of these genetic differences on the cell motility and invasiveness is poorly understood. Here, we report that cellular responses to the mechanical rigidity of the extracellular matrix correlate with the rigidity of the target tissue. We tested a series of single cell populations isolated from MDA-MB-231 breast cancer cell line in a variety of assays where the extracellular matrix rigidity was varied to mimic the environment that these cells might encounter in vivo. There was increased proliferation and migration through the matrices of rigidities corresponding to the native rigidities of the organs where metastasis was observed. We were able to abolish the differential matrix rigidity response by knocking down Fyn kinase, which was previously identified as a critical component of the FN rigidity response pathway in healthy cells. This result suggests possible molecular mechanisms of the rigidity response in the malignant cells, indicating potential candidates for therapeutic interventions

Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells

INTRODUCTION: The metastatic progression of cancer is a direct result of the disregulation of numerous cellular signaling pathways, including those associated with adhesion, migration, and invasion. Members of the Rac family of small GTPases are known to act as regulators of actin cytoskeletal structures and strongly influence the cellular processes of integrin-mediated adhesion and migration. Even though hyperactivated Rac proteins have been shown to influence metastatic processes, these proteins have never been directly linked to metastatic progression. METHODS: To investigate a role for Rac and Cdc42 in metastatic breast cancer cell invasion and migration, relative endogenous Rac or Cdc42 activity was determined in a panel of metastatic variants of the MDA-MB-435 metastatic human breast cancer cell line using a p21-binding domain-PAK pull down assay. To investigate the migratory and invasive potential of the Rac isoforms in human breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed either dominant active Rac1 or dominant active Rac3 into the least metastatic cell variant. Dominant negative Rac1 or dominant negative Rac3 were stably expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1 or Rac3 were analyzed using in vitro adhesion, migration and invasion assays. RESULTS: We show that increased activation of Rac proteins directly correlates with increasing metastatic potential in a panel of cell variants derived from a single metastatic breast cancer cell line (MDA-MB-435). The same correlation could not be found with activated Cdc42. Expression of a dominant active Rac1 or a dominant active Rac3 resulted in a more invasive and motile phenotype. Moreover, expression of either dominant negative Rac1 or dominant negative Rac3 into the most metastatic cell variant resulted in decreased invasive and motile properties. CONCLUSION: This study correlates endogenous Rac activity with high metastatic potential and implicates Rac in the regulation of cell migration and invasion in metastatic breast cancer cells. Taken together, these results suggest a role for both the Rac1 and Rac3 GTPases in human breast cancer progression

Importance of TLR2 on Hepatic Immune and Non-Immune Cells to Attenuate the Strong Inflammatory Liver Response During Trypanosoma cruzi Acute Infection

Trypanosoma cruzi, an obligate intracellular protozoan, is the etiological agent of Chagas Disease that represents an important public health burden in Latin America. The infection with this parasite can lead to severe complications in cardiac, liver and gastrointestinal tissue depending on the strain of parasite and host genetics. Recently, we reported a fatal liver injury in T. cruzi infected B6 mice. However, the local immune response against this parasite is poorly understood. This work highlights some of the molecular and cellular mechanisms involved in liver pathology during the acute phase of infection. Using two mouse strains with different genetic backgrounds and responses to infection, B6 and BALB/c, we found that infected B6 mice develop a strong pro-inflammatory environment associated with high TLR9 expression. Conversely, infected BALB/c mice showed a more balanced inflammatory response in liver. Moreover, higher TLR2 and TLR4 expression were found only in hepatocytes from BALB/c. These data emphasize the importance of an adequate integration of signalling between immune and non-immune cells to define the outcome of infection. In addition, the pre-treatment with TLR2-agonist reverts the strong pro-inflammatory environment in T. cruzi infected B6 mice. These results could be useful in the understanding and design of novel immune strategies in controlling liver pathologies

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2−/−γc−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2−/− γc−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation

An improved method to study NK-independent mechanisms of MTLn3 breast cancer lung metastasis

Toxicolog

Multiple Signaling Pathways are Activated During Insulin-like Growth Factor-I (IGF-I) Stimulated Breast Cancer Cell Migration

In order to display the full metastatic phenotype, the cancer cell must acquire the ability to migrate. In breast cancer, we have previously shown that insulin-like growth factor I (IGF-I) enhances cell motility in the highly metastatic MDA-231BO cell line by activating the type I IGF receptor (IGF1R). This motility response requires activation of IRS-2 and integrin ligation. In order to identify the key molecules downstream of IRS-2, we examined several signaling pathways known to be involved in cell motility. Focal adhesion kinase (FAK) was not activated by IGF-I, but IGF-I caused redistribution of FAK away from focal adhesion plaques. IGF-I treatment of MDA-231BO cells activated RhoA and inhibition of Rho-kinase (ROCK) inhibited the IGF-mediated motility response. The mitogen activated protein kinase (MAPK), p38, was also activated by IGF-I and inhibition of p38 by SB203580 blocked IGF-I induced cell motility. ROCK inhibition with Y-27632 also inhibited p38 phosphorylation suggesting that p38 lies downstream of ROCK. Both Erk1,2 and phosphatidyl-3 kinase (PI3K) were required for IGF-I stimulated cell motility, but only PI3K appeared to be directly downstream of IGF-I. Thus, IGF-I activation of its receptor coordinates multiple signaling pathways required for cell motility. Defining the key molecules downstream of the type I IGF receptor may provide a basis for optimizing therapies directed at this target.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44233/1/10549_2005_Article_4626.pd