An enhanced apoptosis and a reduced angiogenesis are associated with the inhibition of lung colonisation in animals fed an n-3 polyunsaturated fatty acid-rich diet injected with a highly metastatic murine melanoma line.

Both epidemiological and experimental studies indicate that dietary n-3 PUFA inhibit carcinogenesis and tumour growth. Metastatic diffusion has also been found to be affected in animals fed diets containing purified n-3 PUFA or fish oil. In the present study, we investigated whether the metastatic diffusion of a highly metastatic variant (F10-SR cells) isolated from the B16 melanoma F10 line was affected by feeding host animals a diet containing 5 % fish oil. In these animals, compared with those fed a diet containing 5 % maize oil, there was a reduced number of metastatic pulmonary colonies. The immunohistochemical analysis of appropriate markers revealed that the antimetastatic effect of dietary n-3 PUFA was not related to a reduction of proliferation, but rather to an enhanced apoptotic activity. The reduction of von Willebrand factor immunoreactivity found in pulmonary colonies of F10-SR cells grown in fish oil-fed animals indicates that a decrease of angiogenesis contributes to the antimetastatic effect of dietary n-3 PUFA. This conclusion stands in spite of the higher expression of vascular endothelial growth factor observed in pulmonary colonies grown in fish oil-fed animals.

Dietary n-3 polyunsaturated fatty acids enhance metastatic dissemination of murine T lymphoma cells.

Epidemiological investigation and animal studies have shown that dietary n-3 PUFA prevent the development and progression of certain types of cancer. However, conflicting results have been reported by the few studies that focused on the effect of dietary n-3 PUFA on the development of metastases. In the present study, we investigated the metastatic dissemination of murine T lymphoma lines with different metastatic potential transplanted into mice fed a fish oil diet, compared with mice fed a maize oil diet. Transplantation of highly metastatic S11 cells into animals fed a fish oil diet induced a large lymphomatoid infiltration in the spleen, associated with an eight-fold increase in spleen weight, compared with normal animals on the same diet. In contrast, only a limited increase in spleen weight was found in animals transplanted with S11 cells while fed a maize oil diet. No significant increase in spleen weight was found in animals transplanted with low-metastatic 164T2 cells regardless of whether they were fed a fish oil or a maize oil diet. At the end of experiment, an overt cachexia was shown by animals fed a fish oil diet transplanted with S11 cells, but not by those transplanted with 164T2 cells. The particularly high pro-metastatic effect of dietary n-3 PUFA on S11 cells rules out the generalisation that dietary n-3 PUFA inhibit tumour growth and progression.

Tumoral and macrophage uPAR and MMP-9 contribute to the invasiveness of B16 murine melanoma cells.

The aim of this study was to investigate whether tumor cells as well as tumor-associated macrophages (TAMs) contribute to the generation of protease activities essential to tumor cell invasiveness, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9), and the urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). We found that the enhanced invasiveness through Matrigel-coated filters of B16 murine melanoma cells stimulated with IFNgamma was associated with an higher expression of uPAR and MMP-9 in these cells. Moreover, treatment with anti-MMP-9 or anti-uPAR monoclonal antibodies abrogated the increase of invasiveness in IFNgamma-stimulated melanoma cells, suggesting a cooperation of uPA system and MMP-9 in cytokine-stimulated invasiveness. Invasiveness through Matrigel was also enhanced in B16 melanoma cells exposed to a medium conditioned by TAMs, represented in our experimental model by thioglycollate-elicited macrophages co-cultivated with melanoma cells. Macrophages isolated from these co-cultures were found to express higher levels of uPAR and MMP-9 compared to macrophage cultures alone, and the pro-invasive activity of the co-culture-conditioned medium was abrogated by anti-MMP-9 monoclonal antibodies, but not anti-uPAR monoclonal antibodies. Furthermore, the enhanced uPAR and MMP-9 expression in macrophages co-cultivated with tumor cells seems a rather specific phenomenon, generated through a cell-to-cell contact mechanism. On the whole, our data point to a cooperation between tumor cells and macrophages elicited by tumor cells themselves in generating key enzymes essential in the promotion of tumor invasiveness, such as uPAR and MMP-9.

Expression of cyclo-oxygenase-2 in macrophages associated with cutaneous melanoma at different stages of progression.

The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.

Expression of a metastatic phenotype in IFNs-primed/TNFalpha-activated B16 murine melanoma cells: role of JAK1/PKCdelta signal transduction factors.

In previous studies, we found that IFNgamma and TNFalpha generated by activated macrophages stimulate the metastatic potential in F10-M3 cells, a clone isolated from B16-F10 murine melanoma line. In this phenomenon, TNFalpha promoted the expression of a metastatic phenotype in tumor cells previously primed with IFNgamma. Here, we demonstrate that IFNalpha or IFNbeta may replace IFNgamma in priming tumor cells. We also noticed that an enhancement of the expression of p55TNFalpha receptor was associated with the preconditioning of tumor cells with IFNgamma and IFNbeta. By the use of an appropriate inhibitor, we observed that JAK1 signal transduction pathway was involved in the expression of a metastatic phenotype and of p55TNFalpha receptor shown in IFNgamma- and IFNbeta-primed melanoma cells stimulated with TNFalpha. Furthermore, the activity of the protein kinase C (PKC) was required for IFNgamma-primed melanoma cells to express a metastatic phenotype after stimulation with TNFalpha. In conclusion, our study shows that a metastatic phenotype was expressed in B16 murine melanoma cells stimulated with TNFalpha regardless of whether the cells were primed with IFNgamma IFNalpha or IFNbeta. The molecular events leading to the expression of a metastatic phenotype in F10-M3 melanoma cells are represented by: (a) an enhanced expression of p55TNFalpha receptor in IFNs-primed tumor cells dependent on JAK1 signal transduction pathway; and (b) an intact PKC activity during TNFalpha stimulation.

The inhibition of lung colonization of B16-F10 melanoma cells in EFA-deficient animals is related to enhanced apoptosis and reduced angiogenesis.

Previous studies conducted in our laboratory showed that the reproduction of spontaneous and experimental metastases was reduced in host animals deprived of essential fatty acids (EFA). In the present study, we have explored the possibility whether apoptosis, proliferation, and angiogenesis might be involved in the antimetastatic effect of EFA deficiency. To this aim, in pulmonary colonies developed from B16-F10 cells in EFA-deficient animals or in animals fed a 5% corn oil diet, we performed an immunohistochemical analysis of bcl-2/bax proteins, PCNA, and VEGF and von Willebrand Factor (vWF), typical markers of apoptosis, proliferation, and angiogenesis, respectively. Apoptosis was also evaluated by detecting DNA fragments in metastatic cells. We found that the reduction of pulmonary colonies grown in EFA-deficient animals was associated with a high expression of apoptotic activity as revealed by the presence of apoptotic nuclei and a high immunoreactivity for bax. Cell proliferation seemed not to be influenced by EFA deficiency in view of the observation that PCNA was highly expressed in pulmonary colonies of control as well as EFA-deficient animals. Pulmonary colonies developed in EFA- deficient animals showed a lower expression of VEGF and a decreased microvessel density, indicating that a reduced angiogenesis contributes to the antimetastatic effects of EFA deficiency. Our analysis of the results invokes the possibility that a relationship between angiogenesis and apoptosis may account for the diminution of the development of experimental metastases in the lungs of EFA-deficient animals.

Platelet-activating factor (PAF) is the effector of IFN gamma-stimulated invasiveness and motility in a B16 melanoma line.

In this study, we investigated whether PAF synthesized by F10-M3 cells (a clone of B16-F10 melanoma line) mediates the increased capacity of these cells to penetrate into Matrigel upon stimulation with IFN gamma. The determination of PAF synthesized by IFN gamma-stimulated tumor cells revealed that 70% of newly synthesized PAF was released into growth media, while the remaining 30% was associated with the cell bodies. An experimental protocol based on the use of WEB 2086, a PAF receptorial antagonist, was designed to explore which of the two fractions of PAF synthesized by IFN gamma-stimulated F10-M3 cells (released into the growth medium or associated with the cell bodies) is essential to their capacity to migrate through Matrigel. We found that the PAF secreted into growth medium is the fraction responsible for the enhanced invasiveness of melanoma cells stimulated with IFN gamma. We also investigated whether motility of melanoma cells is stimulated by IFN gamma, and, if so, whether PAF is involved in this effect. We found that WEB 2086 prevented the remodeling of stress fibers, examined as an index of cell motility, that we observed in F10-M3 cells stimulated with IFN gamma. Furthermore, the observation that PAF receptor is expressed in IFN gamma-stimulated melanoma cells suggests that the invasive phenotype (e.g. migration through a reconstituted basement membrane and motility) promoted by PAF is based on an autocrine mechanism. On the whole, these results might indicate that PAF contributes to the expression of properties typical of an invasive phenotype in tumor cells stimulated with cytokines.

herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells.

The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy. We report in this article that K(+) ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells. Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.

Enhancement of nitric oxide release in mouse inflammatory macrophages co-cultivated with tumor cells of a different origin.

In the present study we investigated whether synthesis of nitric oxide (NO) by macrophages is affected by contact with tumor cells. Although it is well known that NO generated by macrophages influences different activities related to tumor progression, there is limited information on the modulatory role of tumor cells on NO release by macrophages. The experimental protocol used in our study consisted in the determination of NO secreted by macrophages, either resident or inflammatory, co-cultivated with tumor cells (B16 melanoma and L929 fibrosarcoma cells) at different cell densities and macrophage:tumor cell ratios. This experimental in vitro protocol simulates the different interactions between macrophages and tumor cells that occur during the development of a tumor mass. We found that the co-cultivation with tumor cells induced an increased secretion of NO in macrophages provided that they express an inflammatory phenotype, and they were challenged with LPS or IFNgamma/LPS. Two more variables were found to be critical in the increase of NO generation in inflammatory macrophages cultivated with tumor cells: a high cell density and a prevalence of tumor cells over macrophages. The enhancement of NO secreted in inflammatory macrophages stimulated by tumor cells was not observed in normal murine fibroblasts co-cultivated with tumor cells.

Inhibition of lipoxygenase pathway in macrophages co-cultivated with tumor cells.

Although there is a great deal of interest in the role played by tumor-associated macrophages in tumor progression, the knowledge of the biological mediators involved in the interplay between macrophages and tumor cells is still limited. In the present study, we investigated whether the lipoxygenase pathway in resident murine peritoneal macrophages is affected by contact with tumor cells of a different origin, e.g. murine B16 melanoma and L929 fibrosarcoma cells, and human Hs294T melanoma and HT1080 fibrosarcoma cells. Our experiments have been carried out by using macrophages co-cultivated with tumor cells at different ratios, in order to simulate the relative proportions between macrophages and tumor cells during the in vivo development of a tumor. Reverse phase HPLC analyses of the lipoxygenase products of resident peritoneal macrophages revealed a rather complex profile characterized by a high level of 12(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid followed by leukotriene B(4), 5(S)-hydroxyeicosatetraenoic acid, and lipoxins. Macrophages co-cultivated with tumor cells, both murine and human, showed a marked reduction of lipoxygenase products, mainly in the co-cultures where tumor cells prevailed over macrophages. The characteristic profile of macrophage lipoxygenase products was re-established after removal of tumor cells from the co-cultures. The inhibitory effect on lipoxygenase pathways exerted by tumor cells, was not seen when macrophages were co-cultivated with normal primary murine and human fibroblasts.

Modulation of proteolytic potential and differentiation by CNTF and BDNF in two mouse neuroblastoma clones: relation to invasion.

The effect of CNTF and BDNF on a proteolytic complement instrumental to invasion and on differentiation was studied in two murine neuroblastoma clones, N1 and N7. At the membrane level, gelatinase MMP-2--mainly the activated form--was restrained by CNTF and BDNF to a residual 34% with both factors; membrane-type 1 MMP was down-regulated to 50% (10 h) and 34% (24 h) with both factors; and urokinase-type plasminogen activator was restrained mainly by BDNF to 70%. In the medium, the two gelatinases MMP-2 and MMP-9 were mainly in zymogen form: only MMP-2 was restrained in N1 cells, while only MMP-9 was restrained in N7 cells by both factors, single or in combination. These effects were paralleled by the induction of neurite outgrowth, which was more stimulated in the less differentiated clone. These dose-dependent and transient effects make CNTF and BDNF ideal candidates for constraining the potentially invasive behavior of nervous system tumors.

IFNgamma and TNFalpha account for a pro-clonogenic activity secreted by activated murine peritoneal macrophages.

In the present study, we found that murine peritoneal macrophages elicited by BCG or Listeria monocytogenes release into the media an activity capable of stimulating the lung colonization as well as the expression of MHC class I antigens in B16 melanoma cells. A similar activity has previously been found in media conditioned by Corynebacterium parvum-elicited macrophages. Analysis by gel filtration chromatography of media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages revealed that the material responsible for the pro-clonogenic activity concentrated in chromatographic fractions corresponding to molecular weights (25 to 52 kDa) which are characteristic of certain cytokines. Thus, we challenged the various macrophage-conditioned media with polyclonal antibodies against IFNgamma and TNFalpha, and found that the macrophage pro-clonogenic activity was completely abolished in the presence of anti-IFNgamma antibodies, but only partially inhibited by anti-TNFalpha antibodies. This finding suggests a cooperative participation of the two cytokines to the pro-clonogenic activity of the media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages.

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin.

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.

Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells.

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the "footprints" that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.

hERG1 channels in human esophagus: evidence for their aberrant expression in the malignant progression of Barrett's esophagus.

Ion channels regulate a broad range of cellular activities. Alteration in ion channel function has been reported in different human pathologies, such as cardiac, neuromuscular, autoimmune diseases, and cancer. We investigated the expression of hERG1 K+ channels in the human upper gastrointestinal tract, focusing our attention on the lower esophagus. In particular, we analyzed by both Reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) endoscopic samples obtained from normal subjects, from patients suffering from gastroesophageal reflux, associated or not with esophagitis, and from patients affected by Barrett's esophagus (BE), that is, intestinal metaplasia. None of the normal samples, nor those from patients with gastro-esophageal reflux symptoms and reflux esophagitis expressed the hERG1 protein. On the other hand, 69% of patients with BE expressed hERG1. Since BE is a preneoplastic lesion, dysplasias (Ds) and adenocarcinomas (ADKs) arising on a previously diagnosed BE were also analyzed, and all the samples showed a high expression of the hERG1 protein. The surveillance of patients with BE showed that 89% of those who later developed ADKs displayed hERG1 expression. Data here reported, support the hypothesis that hERG1 expression marks an early step of the progression of normality to cancer in the human esophagus through a metaplastic and dysplastic stage.

Platelet activating factor inhibits the expression of matrix metalloproteinases and affects invasiveness and differentiation in a system of human neuroblastoma clones.

Platelet Activating Factor (PAF), an inflammatory bioactive lipid, has been shown to be involved in the regulation of the activity of matrix metalloproteinases (MMPs). In view of the role played by MMPs in tumor cell invasiveness, we investigated whether PAF influences MMP activity in a system of neuroblastoma clones, the AA5 and AE12 cells, isolated from the human LaN1 neuroblastoma cell line. These clones were characterized by an inverse relationship between invasiveness and differentiative capacity and by the expression of specific cell surface PAF receptors. We found that the levels of mRNAs specific for MMP-2 and for MT1-MMP, the MMP-2 activator, were reduced in both clones treated with 300 nM PAF. These changes are consistent with the reduced secretion and activation of MMP-2 found in the neuroblastoma clones exposed to PAF. These effects were accompanied by an inhibition of invasiveness through Matrigel and by a promotion of differentiation, as revealed by an increased percentage of cells with neurites. The finding that both neuroblastoma clones exposed to the metalloproteinase inhibitors, BB3103 and 1,10-phenanthroline, increased their differentiative capacity and reduced their invasiveness through Matrigel, represents a further indication that PAF modulates differentiation and invasiveness by affecting the activity of MMPs.

Enhanced invasion of hormone refractory prostate cancer cells through hepatocyte growth factor (HGF) induction of urokinase-type plasminogen activator (u-PA).

BACKGROUND: Increased expression of the hepatocyte growth factor (HGF) receptor (MET) is associated with high-grade prostatic adenocarcinoma and metastasis. However, the mechanism through which MET signaling contributes to prostate cancer (CaP) metastasis remains unclear. METHODS: Human PC-3 CaP cells and in vivo selected, isogeneic variant cells of increasing metastatic potential (PC-3M, PC-3M-Pro4, and PC-3M-LN4) were used to investigate the effect of HGF on CaP cell growth, protease production, and invasion. Cell-free urokinase-type plasminogen activator (u-PA) expression and function following HGF treatment were analyzed by Western blot, ELISA, and casein/plasminogen zymography. In vitro invasion stimulated by HGF was measured using Matrigel-coated invasion chambers. RESULTS: Both mRNA and functional protein for MET were detected in each of the CaP cell lines. HGF treatment (0-40 ng/ml) weakly increase proliferation, however, HGF induced soluble u-PA protein and activity 3-fold in the metastatic variant cells. HGF significantly stimulated the invasion of highly metastatic PC-3M-LN4 cells through Matrigel and treatment with specific urokinase receptor inhibitors diminished the HGF-stimulated invasion in a dose-dependent manner. CONCLUSIONS: These results demonstrate the biological significance of u-PA up-regulation in response to HGF in highly metastatic hormone refractory CaP cells.

Ras gene mutations in patients with acute myeloid leukaemia and exposure to chemical agents.

Mutations of the N- and K-ras genes occur in approximately 15-30% of acute myeloid leukaemia patients. The role of the oncogenic ras in leukaemogenesis remains unclear. Few studies have revealed that mutations in the ras oncogene family are more probably found in acute myeloid leukaemia patients with previous exposure to toxic agents. A case-case study was conducted in the areas of Florence and Turin, Italy, to investigate whether the presence of N- and K-ras mutations in acute myeloid leukaemia patients was related to a higher frequency of exposure to chemicals. During a 3-year period, 111 acute myeloid leukaemia patients were enrolled. All the patients were interviewed using a semi-structured questionnaire collecting data on residential history, occupation, personal habits and pathological history. The presence of N- and K-ras mutations was analysed by amplification and synthetic oligonucleotide probes and by the so-called polymerase chain reaction amplification for specific alleles technique. A total of 34 (30.6%) patients were found to harbour ras mutations in N-ras and/or K-ras. Fourteen patients (12.6%) had a single ras mutation and 20 patients (18%) had two ras mutations. A positive association between a priori at risk jobs and ras mutations was found, based on nine exposed cases; the odds ratio, adjusted by age, sex and previous X-ray and/or chemotherapy was 2.8 (95% confidence intervals: 0.9-9.0). When considering only subjects with two ras mutations the odds ratio was 4.8 (95% confidence intervals: 1.2-18.8). The odds ratio for a previous X-ray and/or chemotherapy was 16.2 (95% confidence intervals: 1.8-755.9); when only subjects with two ras mutations were considered, the odds ratio was 26.1 (95% confidence intervals: 2.5-1248.9). In conclusion, our data suggest that ras oncogene mutations might identify a group of leukaemia in people with previous X-ray/chemotherapy or with exposure to chemical agents in the work environment.