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Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 -/- fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27-42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin-rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 -/- fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

The 2-phenylaminopyrimidine derivative STI571 is a selective inhibitor of c-Abl, c-kit, and platelet-derived growth factor-receptor tyrosine kinases and is presently in phase II-III clinical studies. Here, this study reports on a novel pharmacologic activity of the compound, ie, enhancement of the cyto-differentiating, growth-inhibitory, and apoptogenic actions of all-trans-retinoic acid (ATRA). Whereas STI571 is not a cytodifferentiating agent by itself, the compound interacts with ATRA and enhances the myeloid maturation program set in motion by the retinoid in the PML-RARalpha(+) acute promyelocytic leukemia NB4 and the PML-RARalpha(-) myeloblastic HL60 and U937 cell lines. In addition, STI571 relieves the cyto-differentiation block observed in the ATRA-resistant cell lines, NB4.R1, NB4.306, and NB4.007. In NB4 promyelocytes, a RARalpha agonist, but not an RXR agonist, can substitute for ATRA and interact with STI571. By contrast, STI571 is unique among c-Abl-specific tyrosine kinase inhibitors in modulating the pharmacologic activity of ATRA. In NB4 cells, enhanced cyto-differentiation results in increased up-regulation of the expression of a number of genes coding for myeloid differentiation markers, including CD11b, CD11c, and some of the components of the nicotinamide adenine dinucleotide phosphate-oxidase enzymatic complex. All this is accompanied by inhibition of c-Abl tyrosine phosphorylation and retardation of the retinoid-dependent degradation of PML-RARalpha and RARalpha. Stabilization of the 2 retinoic acid receptors is likely to be the result of augmented and accelerated inhibition of the proteasome-dependent proteolytic activity observed on ATRA treatment.

6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RARalpha and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids. (Blood. 2000;95:2672-2682)

The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-gamma agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-gamma or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro-caspase-3 and pro-caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-alpha, which can be completely inhibited by z-VAD.

Although some blood parameters have been suggested to modulate in-vitro induction of sister chromatid exchanges by 1,2:3,4-diepoxybutane (DEB), a metabolite of 1,3-butadiene, the increased sensitivity has largely been assigned to a homozygous deletion of glutathione S-transferase T1 gene (GSTT1 null genotype). However, some DEB-sensitive individuals have been shown to be GSTT1 positive (having at least one undeleted GSTT1 allele). To examine potential causes for this overlap, we evaluated the effect of GSTM1, GSTP1, and GSTT1 genotypes, together with various life-style and blood parameters, on the DEB induction of sister chromatid exchanges and cells with chromosomal aberrations (aberrant cells) in lymphocyte cultures of 115 and 62 human donors, respectively. Our results supported the important role of the GSTT1 genotype in DEB sensitivity; 76% of cultures from GSTT1 null donors but only 4% of those from GSTT1 positive donors were DEB-sensitive, as defined by sister chromatid exchange measurements. The GSTT1 genotype also clearly affected DEB-induced aberrant cells, 92% of GSTT1 null and 8% of GSTT1 positive donors being sensitive to DEB. All individuals showing a high response to DEB in both sister chromatid exchange and aberrant cell analyses were GSTT1 null. Baseline aberrant cell measurements but not sister chromatid exchange measurements were marginally higher among GSTT1 null donors compared with GSTT1 positive donors. GSTM1 and GSTP1 genotypes had no influence on these cytogenetic end-points. Blood transaminases, gamma-glutamyl transferase, urea, creatinine and white blood cell count showed a clear negative association with DEB-induced aberrant cells, whereas wine drinkers had more aberrant cells than non-drinkers. A higher sister chromatid exchange-response to DEB was observed in lymphocytes from women and smokers than from men and non-smokers, respectively. Erythrocyte count correlated negatively with DEB-induced sister chromatid exchanges. Thus, a variety of parameters seemed to modulate the individual DEB-sensitivity together with the GSTT1 genotype. Although the known contributing factors accounted for a considerable part of individual variability in sister chromatid exchanges (59.4%) and aberrant cells (46.7%) in DEB treatment, they did not, however, fully explain the overlap in cytogenetic response between GSTT1 positive and null individuals.

All-trans retinoic acid (ATRA) and interferons (IFNs) are active anticancer agents. ATRA is capable of inducing complete remission in acute promyelocytic leukemia (APL) patients, whereas IFNalpha is successfully used in the treatment of the stable phase of chronic myeloid leukemia. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemias and solid tumors. The molecular basis of these interactions are poorly understood and need to be elucidated. In this review, we summarize a series of recent observations concerning the molecular mechanisms underlying the cross-talk between the intracellular pathways activated by ATRA and IFNs in APL cells. In APL blasts, IFNs regulate the expression of retinoic acid receptors, and ATRA, in turn, modulates the levels and the state of activation of members of the Jak-STAT second messenger pathway. This demonstrates a two-way interaction between ATRA and IFNs, which leads to cross-modulation of genes normally under the control of the retinoid and the cytokine. These data may be relevant in the context of a rational use of the combination between ATRA and IFNs in the clinical management of myeloid leukemias.

We employed the three-way differential staining technique (TWD), which allows SCEs to be distinguished on a per generation basis by scoring third metaphases (M3), in order to study the spontaneous levels of SCEs in normal and high frequency cells (HFCs) that occurred in the first (S1), second (S2) and third (S3) S phases. Fifty one of 900 lymphocytes from 37 healthy donors were defined as HFCs by calculating the 95th percentile of the distribution of SCEs in S1 + S2. 'Normal' cells presented almost the same number of SCEs after the first, second and third cell cycles (SCE averages of 2.43, 2.04 and 3.53 respectively). In contrast, HFCs showed a higher SCE count in S1, which decreased rapidly through the cycles and reached baseline level at S3 (SCE averages of 7.18, 4.29 and 3.45 respectively). This would suggest that the lesions responsible for the higher SCE frequency in HFCs were effectively removed after two cell cycles and strongly support the hypothesis that HFCs are lymphocytes which accumulate higher levels of DNA lesions through time.

Peripheral blood lymphocytes from a sample of 62 randomly selected donors were analysed for spontaneous and diepoxybutane (DEB)-induced chromosomal aberrations (CA). These individuals were part of a larger sample of 122 subjects whose DEB responsiveness was evaluated by means of sister chromatid exchange (SCE) analysis. Confounding factors (such as smoking, wine and coffee consumption, occupation and haematological factors) were analysed for their effect on individual DEB-responsiveness, but no statistically significant associations were observed. Interestingly, a bimodal distribution of aberrant cell frequencies was clearly detectable, showing the existence of DEB-sensitive subjects belonging to the second mode (CA frequencies > 19%). When responsiveness evaluated by means of CA induction was compared with SCE responsiveness, it was noted that all SCE-inducible subjects (> 110.9 SCEs/cell) belonged to the second mode of CA frequency distribution. On the other hand, highly CA inducible individuals did not necessarily show a higher SCE-response, although their DEB-induced SCE frequencies were above average (92 SCEs/cell). DEB-induced CA frequency correlated with baseline levels, indicating that DEB-sensitive individuals also showed higher spontaneous chromosome damage (3.6 versus DEB-resistant 2%, P < 0.05). Finally, when simple and multiple regression analyses were carried out, DEB-sensitivity appeared negatively related to haematic concentrations of proteins and uric acid (intercept 0.131 +/- 0.011, slope -0.029 +/- 0.0116, r = -0.39; P < 0.01), probably due to its antioxidant activity. This finding confirmed previous observations on the scavenger activity of plasma factors on DEB mutagenicity.

As humic compounds are naturally widespread in the environment and present in surface water, studies on their genotoxicity are justified. Humic acid (HA) has not been demonstrated to be genotoxic either in vitro or in vivo. In the present paper we investigated its activity both in intestinal and bone marrow cells following a single dose (100 mg/kg b.w. corresponding to 0.5 ml per animal of an aqueous solution of 4 g/l) of HA administered to mice by gastric intubation, to mimic the most likely route of human exposure. HA induced structural and, in particular, numerical chromosome abnormalities in intestinal cells. A marginal, non-significant induction of aneuploidy was also found in bone marrow cells.

Two coal-derived humic substances (Sulcis and South Africa, Eniricerche, Italy) have been evaluated for their mutagenic activity on TA98 and TA100 Salmonella typhimurium strains, either in presence or in absence of metabolic activation (S9). Both compounds showed no effect on the two strains, as observed with natural humic acid (Fluka). After chlorination, coal-derived humic acids induced a strong dose-related increase in the number of revertants on TA100 without S9, whose extent was directly proportional to the chlorination ratios. Such effect was completely suppressed when a sodium thiosulfate solution (10%) was added at the end of the chlorination period (about 90 h). The analogies with natural humic acid mutagenicity are discussed.

Spontaneous and diepoxybutane (DEB)-induced sister-chromatid exchanges (SCEs) were examined in whole-blood lymphocyte cultures of 3 men and 4 women. A strong increase in mean number of SCEs per cell with increasing DEB concentrations (0, 2, and 4 microM) was observed in cultures of all subjects, but 3 of the donors were clearly more sensitive than the others. The SCE measurements were repeated 2-6 times per donor over a period of 55 months to assess the stability of the individual SCE response. The results showed that SCE induction by DEB was steady in the individuals during the follow-up at each DEB dose, with no significant differences among the repeated experiments. At 4 microM DEB, the DEB-sensitive and -resistant donors could be reliably be differentiated from each other in all trials. As DEB-sensitivity has been suggested to be due to the lack of glutathione S-transferase (GST) T1, the donors were genotyped for the presence of GSTT1 and GSTM1 genes. The 3 individuals found to be DEB-sensitive were all of the GSTT1 null genotype, whereas the 4 DEB-resistant donors were GSTT1 positive, which supported the role of the GSTT1 gene in determining DEB-sensitivity. Three of the DEB-resistant and none of the DEB-sensitive had the GSTM1 null genotype. Thus, the lack of the GSTM1 gene was not associated with the DEB-sensitivity trait. In conclusion, the present findings show that individual SCE responses to treatment of cultured human lymphocytes with DEB can reliably be reproduced in repeated trials. The results confirm that the GSTT1 gene but not the GSTM1 gene is important in determining individual sensitivity to the in vitro genotoxicity of DEB.

Liquid holding (LH) recovery was matched with three-way differential staining (TWD) to assess the reduction of damage induced in DNA following treatment with 4-nitroquinoline-1-oxide (4NQO) in resting (GO) lymphocytes. Human peripheral lymphocytes (HPL) from three donors were used to evaluate lesion persistence and individual repair capacity. Our data are in contrast to those for diepoxybutane (Ponzanelli et al., 1995) and suggest that LH recovery is completely inefficient in removing 4NQO induced lesions, which are only partially repaired after one cell cycle.

Previous authors investigated individual responsiveness to mutagens by assessing cytogenetic damage following in vitro treatment. Diepoxybutane (DEB) has been used to assess chromosome instability both in repair-deficient and normal subjects. Since bimodal distribution of sister chromatid exchanges (SCEs) or chromosomal aberration (CAs) frequencies has been observed in normal subjects, we investigated the possible factors determining the 'high-respondent' phenotype. The bimodal-shaped distribution suggested the presence of a single factor responsible far this phenotype. Our data showed that red blood cells are involved in determining the sensitivity of lymphocytes to DEB induced SCE. The existence of a polymorphic factor in red cells involved in DEB detoxification is suggested.

Many studies on DNA repair mechanisms in mammalian cells have used liquid holding (LH) recovery to evaluate premutational damage repair. We used human peripheral lymphocytes (HPL) to assess damage reduction during the G0 phase. This technique was matched with the three-way differential (TWD) staining that allows identification of sister-chromatid exchanges (SCE) per cell cycle in third metaphases. By adopting this approach, the persistence of diepoxybutane (DEB)-induced lesions during subsequent cycles and individual repair capacity in LH conditions were measured. Our results show that most DEB-induced damage was repaired during the first cell cycle; a large part of lesions were removed during LH recovery, demonstrating G0 HPL repair capacity.