Radon progeny exposure and lung cancer risk among non-smoking uranium miners.

Studies of miners provide the basis for public health efforts to reduce residential radon progeny exposure. Because the preponderance of households do not have members who smoke indoors, studies of non-smoking miners contribute essential data for risk assessments for residential radon progeny exposure. We studied a cohort of 2,209 never-smokers who were underground uranium miners employed in the western U.S. from 1956 to the early 1990's and who participated in a screening program for lung cancer conducted by Saccomanno and colleagues. After determining the vital status and cause of death in the cohort, we conducted a nested case-control study of 55 lung cancer deaths in males and 3 age-matched controls for each case. The relative risk of lung cancer was 29.2 (95% CI 5.1, 167.2) for miners with greater than 1,450 WLM compared with those exposed to less than 80 WLM. Temporal factors affected risk, including average dose rate, which was inversely associated with lung cancer risk, and the length of time since last exposure, which was directly associated with decreased risk. As in studies of non-smokers and smokers combined, the exposure response relationship in never-smokers was consistent with a decreased slope at higher WLM, which resulted, in part, from an inverse dose rate effect.

Errors and biases in the diagnosis of cancer of the lung and their influence on risk estimates.

When diagnosed as primary lung cancer, metastases from the abdomen, plus false negative cases have little effect on epidemiology studies of male smokers, but may result in a severe dilution of the lung cancers among women and nonsmokers. We have attempted to quantitate this handicap for epidemiological studies using two approaches. The relative frequency of diagnosed primary lung and abdominal cancer among males, women, and nonsmokers differs substantially and is used here to calculate magnitude. The second approach postulates that the ratio of nonsmokers among persons with squamous cell lung cancer and primary adenocarcinoma of the lung would be constant by sex if there were no distortion by abdominal metastases. These two approaches indicate that the much higher ratio of metastatic disease diagnosed as primary lung cancer among nonsmoking women (factor of 15 to 20), makes it more difficult to identify an environmental carcinogen among women or nonsmokers than among male smokers in case-control studies.

Chronic diffuse interstitial fibrosis of the lung in uranium miners.

Many uranium miners have been disabled by and died of pulmonary fibrosis that was not recognized as an occupational disease. A review of animal studies, complications from whole body irradiation, pulmonary function, and mortality studies of uranium miners led us to suspect radiation-induced chronic diffuse interstitial fibrosis in miners who had inhaled excessive radon progeny. A selected group of uranium miners (22) with severe respiratory disease (but no rounded nodules in chest films) were studied. Lung tissue from five disclosed severe diffuse interstitial fibrosis, with "honeycomb lung" in all. Some also had small anthrasilicotic nodules and birefringent crystals. Although quartz crystals probably contributed, we concluded that the predominant injurious agent in these cases was alpha particles from radon progeny. This disease, after a long latent period, usually results in pulmonary hypertension, shortness of breath, and death by cardiopulmonary failure.

The LIM protein RBTN2 and the basic helix-loop-helix protein TAL1 are present in a complex in erythroid cells.

Chromosomal translocations in T-cell acute leukemias can activate genes encoding putative transcription factors such as the LIM proteins RBTN1 and RBTN2 and the DNA-binding basic helix-loop-helix transcription factor TAL1 associated with T-cell acute lymphocytic leukemia. While not expressed in normal T cells, RBTN2 and TAL1 are coexpressed in erythroid cells and are both important for erythroid differentiation. We demonstrate, using anti-RBTN2 and anti-TAL1 antisera, that the LIM protein RBTN2 is not phosphorylated and is complexed with the TAL1 phosphoprotein in the nucleus of erythroid cells. A complex containing both RBTN1 and TAL1 also occurs in a T-cell acute leukemia cell line. Since both RBTN2 and TAL1 are crucial for normal erythropoiesis, these data have important implications for transcription networks therein. Further, since both proteins can be involved in leukemogenesis, these data provide a direct link between proteins activated by chromosomal translocations in T-cell acute leukemia.

Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.

Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron.

The structure of LIM domains has major implications for transcription because proteins such as Is1-1 contain two LIM domains associated with a homeodomain, and RBTN1/Ttg-1 and RBTN2/Ttg-2 contain two LIM domains but no homeodomain. Conserved cysteine and histidine residues in the LIM domains suggest a metal-binding role. RBTN and Is1-1 LIM proteins have been made in Escherichia coli and insect cell expression systems and their metal content has been determined using atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy. LIM proteins expressed in soluble form contain zinc atoms, whereas bacterial inclusion bodies invariably also have Fe-S clusters. The latter are identified as linear [Fe3S4]+ clusters and appear to result from incorrect metal coordination by E. coli. These studies show that RBTN1, RBTN2, and Is1-1 are metalloproteins that contain zinc but not iron and, therefore, that the LIM domain represents a zinc-binding domain.

Nuclear factor of activated T cells contains Fos and Jun.

The nuclear factor NF-AT (ref. 1) is induced in T cells stimulated through the T-cell receptor/CD3 complex, and is required for interleukin-2 (IL-2) gene induction. Although NF-AT has not been cloned or purified, there is evidence that it is a major target for immunosuppression by cyclosporin A (CsA) and FK506 (refs 2-7). NF-AT induction may require two activation-dependent events: the CsA-sensitive translocation of a pre-existing component and the CsA-resistant synthesis of a nuclear component. Here we report that the newly synthesized nuclear component of NF-AT is the transcription factor AP-1. We show that the inducible nuclear form of NF-AT contains Fos and Jun proteins. Furthermore, we identify a pre-existing NF-AT-binding factor that is present in hypotonic extracts of unstimulated T cells. On the basis of binding, reconstitution and cotransfection experiments, we propose that activation of NF-AT occurs in at least two stages: a CsA-sensitive stage involving modification and/or translocation of the pre-existing NF-AT complex, and a CsA-insensitive stage involving the addition of newly synthesized Fos or Fos/Jun proteins to the pre-existing complex.

The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter.

Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.

Analysis of the AP-1 sites in the IL-2 promoter.

We have investigated the role of the two AP-1 sites, located at approximately -150 and -180 bp relative to the transcription start site, in induction of the IL-2 promoter through the TCR/CD3 complex. We show that only the proximal (-150 bp) AP-1 site is functional in vitro, as judged by its ability to bind nuclear proteins from T cells stimulated with Ag or anti-CD3 epsilon. The inducible nuclear proteins binding to this site have the characteristics of AP-1, as judged by their kinetics of induction, the ability to compete and be competed efficiently by a metallothionein AP-1 site oligonucleotide, and their reaction with antibodies to Fos and Jun proteins. Mutations in the proximal AP-1 site greatly diminish or abrogate induction of the IL-2 promoter, indicating that the site is also functional in vivo. Although the distal (-180 bp) AP-1 site is incapable of direct binding to nuclear proteins from activated T cells, a mutation in this site diminishes IL-2 promoter induction, suggesting that this site may also be functional in vivo. Cotransfection of a 5' IL-2-chloramphenicol acetyltransferase plasmid with c-Fos and/or c-Jun enhances the induction of IL-2-chloramphenicol acetyltransferase activity, confirming that the IL-2 promoter contains a functional AP-1 site. Both AP-1 sites may be targets for c-Fos action, as inferred from the results of experiments in which c-Fos was cotransfected with internal deletion mutants of the IL-2 promoter lacking either AP-1 site. Northern analysis indicates that mRNAs for at least six members of the Fos/Jun family (c-fos, fosB, fra-1, c-jun, junB, and junD) are expressed in activated Ar-5 cells; thus the AP-1 sites of the IL-2 promoter may bind different dimeric Fos/Jun complexes at different times after T cell activation, perhaps mediating both positive and negative regulation of the IL-2 promoter.

Transformation of T lymphocytes by the v-fos oncogene.

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.

Air pollution and fatal lung disease in three Utah counties.

A unique situation found in two Utah counties has made it possible to estimate the fraction of respiratory cancer and nonmalignant respiratory disease (NMRD) deaths, which are attributable to community air pollution (CAP) in one county. The two counties were very similar in many ways, including low smoking rates, until a steel mill constructed during WW II caused substantial CAP in one of them. Subsequent differences in mortality rates from both respiratory cancer and NMRD are striking. A third county, similar to many counties outside Utah, was included in the analysis for comparison. In one county, 30-40% of the respiratory cancer and NMRD deaths were attributable to CAP. In this county, NMRD deaths (but not respiratory cancer deaths) were slightly more frequent than in Salt Lake County where smoking rates were twice as high.