04-822 Sigma-AldrichAnti-Rho (-A, -B, -C) Antibody, clone 3L74, rabbit monoclonal

PURPOSE: Rho family members are small GTPases that are known to regulate malignant transformation and motility of cancer cells. The activities of Rhos are regulated by molecules such as guanine nucleotide dissociation inhibitors (GDIs). This study determined the levels of expression and the distribution of Rho-A, -B, -C, and -G, and Rho-6, -7, and -8, as well as Rho-GDI-beta, and Rho-GDI-gamma, in breast cancer and assessed their prognostic value. EXPERIMENTAL DESIGN: The distribution and location of Rhos and RhoGDIs were assessed using immunohistochemical staining of frozen sections. The levels of transcripts of these molecules were determined using a real-time quantitative PCR. Levels of expression were analyzed against nodal involvement and distant metastasis, grade, and survival over a 6-year follow-up period. RESULTS: The levels of Rho-C, Rho-6, and Rho-G were significantly higher in breast cancer tissues (n = 120) than in background normal tissues (n = 32). However, the level of Rho-A and -B and rho-7 and -8 was found to be similar in tumor and normal tissues. Immunohistochemical staining revealed the high level of staining of Rho-C protein in tumor cells. The levels of Rho-GDI-gamma transcripts were found to be significantly lower in tumor tissues than in normal tissues (P < 0.05 and P < 0.001, respectively). Node-positive tumors have significantly higher levels of Rho-C and Rho-G, and lower levels of Rho-GDI and Rho-GDI-gamma transcripts, than do node-negative tumors. Significantly higher levels of Rho-C and Rho-G were seen in patients who died of breast cancer than in those who remained disease free. Patients with recurrent disease, with metastasis or who died of breast cancer, also exhibited higher levels of Rho-6 but lower levels of Rho-GDI-gamma. Higher-grade tumors were also associated with low levels of Rho-GDI and Rho-GDI-gamma. CONCLUSIONS: Raised levels of Rho-C, Rho-G and Rho-6 and reduced expression of Rho-GDI and -GDI-gamma in breast tumor tissues are correlated with the nodal involvement and metastasis. This suggests that the expression of Rhos and Rho-GDIs in breast cancer is unbalanced and that this disturbance has clinical significance in breast cancer.

The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases, which are likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases, p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. Here, we describe the purification of a novel Rho-associated kinase, p140, which appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing revealed that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases, which are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase promotes kinase activity substantially, and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases that mediates their effects on rearrangements of the actin cytoskeleton.

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways.