Juan Carlos Lacal

Rho-regulated signals induce apoptosis in vitro and in vivo by a p53-independent, but Bcl2 dependent pathway

Rho proteins are a branch of GTPases that belongs to the Ras superfamily which are critical elements of signal transduction pathways leading to a variety of cellular responses. This family of small GTPases has been involved in diverse biological functions such as cytoskeleton organization, cell growth and transformation, cell motility, migration, metastasis, and responses to stress. We report that several human Rho proteins including Rho A, Rho C and Rac 1, are capable of inducing apoptosis in different cell systems like murine NIH3T3 fibroblasts and the human erythroleukemia K562 cell line. Since K562 cells are devoid of p53, apoptosis induced by Rho in this system is independent of p53. Rho-dependent apoptosis is mediated by the generation of ceramides, and it is drastically inhibited by ectopic expression of Bcl2, both under in vitro and in vivo conditions. Furthermore, the human oncogenes vav and ost that have been shown to function as guanine exchange factors for Rho proteins, were also able to induce apoptosis under similar conditions. Finally, we also report that the levels of endogenous Rho proteins are increased when U937 myeloid leukemia cells are exposed to apoptosis-inducing conditions such as TNFα treatment. Furthermore, TNFα-induced apoptosis in these cells is inhibited by expression of a dominant negative mutant of Rac 1 but it is not affected by a similar mutant of Rho A. These results suggest that Rho proteins play an important role in the physiological regulation of the apoptotic response to stress-inducing agents.

The Ras family of GTPases in cancer cell invasion.

Abstract. The ability of tumoral cells to invade surrounding tissues is a prerequisite for metastasis. This is the most life-threatening event of tumor progression, and so research is intensely focused on elucidating the mechanisms responsible for invasion and metastasis. The Ras superfamily of GTPases comprises several subfamilies of small GTP-binding proteins whose functions include the control of proliferation, differentiation, and apoptosis, as well as cytoskeleton organization. The development of metastasis is a multistep process that requires coordinated activation of proliferation, motility, changes in normal cell-to-cell and cell-to-substrate contacts, degradation of extracellular matrix, inhibition of apoptosis, and adaptation to an inappropriate tissue environment. Several members of the Ras superfamily of proteins have been implicated in these processes. The present review summarizes the current knowledge in this field.

Screening for new compounds with antiherpes activity

A number of compounds have been tested for antiherpes activity. Actinobolin, amicetin, carrageenan, laspartomycin, megalomycin C, pleuromutilin, suramin and tetracenomycin C showed significant protection of HeLa cell monolayers infected with herpes simplex virus type 1. The action of these new antiherpes compounds was compared with those antiherpes agents that have been described previously. Actinobolin, amicetin and tetracenomycin C were also active against viruses other than herpes simplex.

Modification of Membrane Permeability in Poliovirus-infected HeLa Cells: Effect of Guanidine

A drastic modification in permeability to monovalent ions occurred in HeLa cells infected with poliovirus, starting in the period from the third to the fourth h post-infection. The bulk of poliovirus protein synthesis took place from the third to the sixth h in cells in which the concentration of monovalent ions in the cytoplasm had changed considerably compared to uninfected cells. Under our conditions of infection (HeLa cells grown in monolayer), poliovirus translation continued beyond the eighth h. Modification of permeability to 86Rb+ ions induced by poliovirus infection was prevented by the addition of 2 mM-guanidine at the time of infection. However, when poliovirus replication was allowed to take place for 1 h before addition of guanidine, the membrane did become modified to some extent. The degree of leakiness to 86Rb+ ions increased when guanidine was added later. The blockage of membrane leakiness by early addition of guanidine was overcome by the addition of choline. The inhibition of cellular protein synthesis, which occurred early in infection or in the presence of guanidine, did not coincide with the modification of permeability to 86Rb+ ions. Viral protein synthesis was necessary in order to modify the membrane late in infection, since addition of 10(-5) M-cycloheximide during the first 2 h of infection prevented the leakiness to 86Rb+ ions observed from the fourth h after infection. Membrane potential, as measured by the lipophilic cation TPP+ (tetraphenylphosphonium), dropped from the fourth h post-infection. This change in the membrane was also prevented when viral gene expression was inhibited by the presence of guanidine.

Biological Function of Aplysia californica rho Gene

rho genes are a family of genes which are structurally related to the oncogenic ras family. The primary structure of rho genes has been elucidated for the marine snail Aplysia californica, two S. cerevisiae genes, and three human versions, rho A, B and C. They all codify for proteins of an approximate M.W. of 21 kDa (rho-p21) which show 35% homology to the ras proteins. It has been observed that rho proteins are ADP-ribosylated by the botulinum toxin C3 exoenzyme, suggesting that rho proteins could be involved in regulating neuronal function. However very little is known about their actual biological functions. While the human rho A and rho C products have been related to cytoeskeleton organization, the rho A product has a weak transforming activity. We have investigated the biological properties of the Aplysia californica rho-p21 protein when introduced into an heterologous system, and found that it does not induce foci in a regular NIH-3T3 transfection assay. However, the morphology of the cells was slightly altered and cells grew to higher cell densities. Moreover, transforming activity was detected when isolated cell lines were inoculated into nude mice. To further investigate the potential transforming activity of the Aplysia gene, we have also generated a Gly→Val mutation at position 14, equivalent to the activating mutation found in oncogenic ras genes. No apparent increase in the transforming activity was observed, indicating that the effects on growth behaviour are probably not the primary function of rho proteins.