Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer.

AIMS: Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a post synaptic density-95/Disk-large/ZO-1 homologous domain-containing protein that is involved in the linkage of integral membrane proteins to the cytoskeleton and plays an important role in cell signalling. To gain insights into its biological relevance, this study examined expression of EBP50 in two cohorts of breast carcinoma. METHODS AND RESULTS: Forty-nine breast carcinoma tissue specimens were first examined by both immunohistochemistry and RNA in situ hybridization. EBP50 expression was correlated with various clinicopathological variables. The relative abundance of EBP50 mRNA in breast carcinomas and their corresponding normal tissue was compared using reverse transcriptase-polymerase chain reaction (RT-PCR). EBP50 immunoreactivity was then further independently validated in 120 breast carcinomas on tissue microarrays. EBP50 immunoreactivity was observed in morphologically normal and cancerous epithelial cells contrasting with the adjacent immunonegative stromal cells. An elevated cytoplasmic accumulation of EBP50 protein was readily detected in 73.5-80% of breast carcinomas. EBP50 immunoreactivity was significantly associated with tumour stage, lymph node and oestrogen receptor status. These immunohistochemical observations were further validated using RNA in situ hybridization and RT-PCR. EBP50 immunoreactivity was significantly correlated with the mRNA expression level. CONCLUSION: Oestrogen-responsive EBP50 may play an important role in tumour progression and might be a potential marker of invasiveness for breast cancer.

Increased fatty acid synthase is a therapeutic target in mesothelioma.

Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.

c-Myc suppresses the tumorigenicity of lung cancer cells and down-regulates vascular endothelial growth factor expression.

The c-myc oncogene is frequently amplified in cells grown from lung tumors and has been linked to the malignancy of these cancers. In support of this, c-myc transfection enhances the in vitro proliferation and soft agar cloning of human small cell lung cancer (SCLC) cells. In this study, we surprisingly found that c-myc expression suppressed the formation of tumors by SCLC cells in athymic nude mice. c-myc expression down-regulated the protein and transcript for vascular endothelial growth factor (VEGF) in these SCLC cells, as well as VEGF transcript in rat fibroblasts manipulated for c-myc expression and in liver cells of c-myc-transgenic mice. Finally, bivariate and multivariate analyses demonstrated that the probability of tumor formation from lung cancer cell lines was negatively correlated with the relative expression of c-Myc, positively correlated with the relative expression of VEGF, and that the latent time to tumor formation was increased by the expression of c-Myc and decreased by the expression of VEGF. We hypothesize that, for lung cancer cells, c-Myc suppresses the formation of tumors in vivo by down-regulating VEGF, and that the amplification of c-myc seen in cells grown from lung tumors with a poor prognosis is an artifact of selection for growth in vitro.

Alternative splicing in 5'-untranslational region of STK-15 gene, encoding centrosome associated kinase, in breast cancer cell lines.

Centrosomes maintain genomic stability by establishing the bipolar spindles during cell division and, execute accurate segregation of chromosomes during mitosis. In this study, we have demonstrated that there are three forms of STK-15 gene in breast cancer cell lines. Alternative splice positions are located in 5'-untranslated region of STK15 gene. The results of in vitro translation experiments revealed that the alternative splicing in the 5'-untranslated region of STK15 had no effect on protein translation. The differential expression patterns of these alternatively spliced STK15 in breast cell lines and primary tumors therefore suggest that STK15 gene transcription may be differentially regulated or stabilized in these cells.

The induction of spermidine/spermine N1-acetyltransferase (SSAT) is a common event in the response of human primary non-small cell lung carcinomas to exposure to the new antitumor polyamine analogue N1,N11-bis(ethyl)norspermine.

Several new polyamine analogues have been developed for the treatment of human solid tumors. The phenotype-specific activity of some of these analogues has been associated with the superinduction of the rate-limiting enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Using immunohistochemistry, we found a majority (64%) of human primary lung cancer explants to exhibit high expression of SSAT after treatment with 10 microM N1,N11-bis(ethyl)norspermine, an agent currently undergoing Phase II clinical trials against several important human solid tumors. The staining of SSAT was found specifically in the tumor tissue and not in the neighboring normal lung tissue. These results demonstrate the ability to detect induction of SSAT in clinical specimens and suggest the potential of this test as a prognostic indicator of drug response.

A novel cytoplasmic substrate for cdk4 and cdk6 in normal and malignant epithelial derived cells.

Cyclins and cyclin-dependent kinases (cdk) have been identified as important regulators of cell replication. Molecular alteration in the cdk pathways appear to be important in cancer with some cyclins (eg cyclin D and E) proposed to be oncogenes and some inhibitors of cdk (eg p16) proposed to be tumor suppressor genes. In human breast carcinoma cell line MDA361 both cyclin D and E are overexpressed and cdk 4 and 6 are the predominate kinases which phosphorylate retinoblastoma protein and to a greater extent a novel 88 kDa protein. This 88 kDa protein was detected as a significant substrate in five of seven breast carcinoma cell lines, three lung carcinoma cell lines as well as in primary breast and lung epithelium. Normal human and murine T lymphocytes and established lymphoid cell lines are devoid of this protein and minimal amounts were detected in normal human fibroblast. In contrast to retinoblastoma protein, the 88 kDa protein appears to be more prevalent in the cytosolic than the nuclear subfraction. The phosphorylation of this 88 kDa protein by the G1 associated cdks suggest that this protein may represent another targeted substrate regulating the G1 phase of the cell cycle.

Spontaneous hemothorax caused by a chest wall chondrosarcoma.

A 48-year-old man who presented with chest pain was found to have a spontaneous hemothorax caused by a large grade II chondrosarcoma. This was diagnosed on the basis of the findings from thoracotomy. After chest tube drainage of the hemothorax, the tissue obtained by two computed tomography-guided biopsies of a residual mass did not yield findings that allowed diagnosis of the tumor. This unusual case illustrates the importance of a systematic workup whenever a spontaneous hemothorax is encountered.

Comparison of the toxic effects of hydrogen peroxide and ozone on cultured human bronchial epithelial cells.

In this study, we compared the cytotoxic and genotoxic effects of hydrogen peroxide and ozone on cultured human airway epithelial cells in primary culture. Both agents caused a dose-dependent loss in the replicative ability of epithelial cells and at higher levels of exposure caused acute cytotoxicity as measured by release of lactate dehydrogenase. Differences were seen, however, between the agents' effects with regard to induction of DNA single strand breaks as measured by alkaline elution:; whereas single-strand breaks were detected in significant amounts at concentration of hydrogen peroxide that cause acute cytotoxicity, none were detected at any of the levels of ozone exposure examined. A difference was also seen in the ability of the iron chelator deferoxamine to protect cells from the effect of the two oxidants. Preincubation of cultures with deferoxamine appreciably attenuated the toxicity of hydrogen peroxide but not of ozone. These data suggest that ozone has significant toxic effects on bronchial epithelial cells not mediated through the generation of hydrogen peroxide or hydroxyl radical. Furthermore, the data indicate that the inhibiting action of ozone on cell replicative ability is not mediated through a mechanism related to DNA single strand breaks.

Physiologic modulation of bronchial epithelial cell barrier function by polycationic exposure.

Bronchial epithelial cells provide a functional barrier to the movement of water and solutes between the luminal and interstitial compartments of the lung. Barrier integrity can be compromised by a variety of factors, including polycationic proteins released by inflammatory cells. We investigated the characteristics of epithelial barrier function and its modulation by cationic stimuli in canine bronchial epithelial (CBE) cells grown in culture. Morphologic characteristics were examined, and barrier function was assessed by measurements of transepithelial mannitol flux (flux) and electrical resistance (RT) during a stable, 3- to 14-day culture period. CBE cultures exhibited progressive mucociliary differentiation and contained nonciliated, ciliated, and neutral and acidic mucin-secretory cells. The synthetic polycation, poly-L-lysine (PLL), from 2.5 to 10 micrograms/ml, caused dose-related increases in flux and decreases in RT that were not accompanied by detectable release of lactate dehydrogenase (LDH) or changes in histochemical appearance. The effect on RT spontaneously reversed over a 15-h recovery period. The action of PLL on flux was not attenuated by treatment of the cells to stabilize cytoskeletal contractile elements but was immediately attenuated by the addition of heparin to the challenged cells. These results indicate that modulation of the barrier integrity of bronchial epithelial cells by cationic proteins, such as those released by inflammatory cells, represents a physiologic process that may be regulated by endogenous anionic factors.

Immunohistochemical staining of human spermidine/spermine N1-acetyltransferase superinduced in response to treatment with antitumor polyamine analogues.

A superinduction of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT) accompanies the phenotype-specific cytotoxic response to a class of antitumor polyamine analogues in several important human solid tumor models. A highly specific antiserum against the human SSAT protein has been developed. Using this antiserum we demonstrate that polyamine analogue treatment in vitro or in vivo results in an induction of SSAT protein which is uniformly distributed throughout the cytoplasm of treated tumor cells. This new biochemical tool will be useful in the examination of the association of superinduced SSAT activity and cell type-specific cytotoxicity. Additionally, since clinical trials have begun on one of the SSAT-inducing polyamine analogues, this antiserum may be useful as a diagnostic tool in differentiating responsive and nonresponsive tumor cell populations in treated patients.

Right and left ventricular cultured endocardial endothelium produces prostacyclin and PGE2.

The endothelium profoundly affects subjacent vascular smooth muscle function. An analogous relationship between endothelial endocardial cells (EEC) and the myocardium is suggested by Brutsaert et al.'s observation that EEC modulate the contractility of subjacent myocardium. Prostanoids are a major product by which vascular endothelium affects smooth muscle, but similar prostanoid production by EEC has not been described. To determine whether both right and left ventricular EEC produce prostacyclin (PGI2) and prostaglandin E2 (PGE2), ovine EEC were cultured. EEC prostanoid production was measured under basal conditions and after stimulation with arachidonic acid or calcium ionophore A23187. EEC from both ventricles demonstrated sustained prostacyclin and PGE2 production. Prostacyclin production was 10 times greater than PGE2. These results suggest that endocardial prostanoid production could act both locally, to modulate platelet and myocardial function, and distally, on downstream vascular tone.

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Measurement of neutrophil activation and epidermal cell toxicity by palytoxin and 12-O-tetradecanoylphorbol-13-acetate.

Palytoxin is a human and mouse skin irritant and complete mouse skin tumor promoter that differs from the well-studied phorbol ester class of tumor promoters in many respects. In this study, we have found palytoxin to stimulate the production of superoxide by isolated human neutrophils using electron paramagnetic resonance (EPR) spectroscopy with the spin-trap DMPO. This stimulation of oxyradical production by palytoxin is relatively weak, however, when compared to that by 12-O-tetradecanoylphorbol-13-acetate (TPA). The maximal amount of oxyradicals produced by palytoxin-stimulated neutrophils is 10(-4) mumols/10(6) neutrophils, and this stimulation requires nanomolar concentrations of palytoxin, with half maximal stimulation at concentrations of approximately 30 nM. In contrast, the tumor promoter TPA causes human neutrophils to generate in excess of 10(-3) mumols oxyradicals/10(6) neutrophils with concentrations as low as 1 nM. Toxicity to cultured human epidermal cells was observed at very low concentrations of palytoxin, with 50% loss of colony-forming efficiency observed at approximately 3 x 10(-13) M. For TPA, 50% loss of colony-forming efficiency for cultured epidermal cells requires approximately 5 nM. Thus, although palytoxin stimulates superoxide production in isolated neutrophils, epidermal cells are sensitive at much lower concentrations and are likely to be the important target cell in vivo. This is in contrast to TPA, where neutrophils are stimulated at concentrations less than those required to produce pathological effects on epidermal cells, suggesting that neutrophils may be an important target cell for TPA in vivo.

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.

Human mesothelioma cells and asbestos-exposed mesothelial cells are selectively resistant to amosite toxicity: a possible mechanism for tumor promotion by asbestos.

To determine if asbestos exposure could contribute to mesothelial cell carcinogenesis by selection and/or expansion of an initiated cell population, we compared normal human pleural mesothelial cells to either human mesothelioma cell lines or mesothelial cells transfected with cancer-related genes for sensitivity to amosite fibers in vitro. Neither normal nor mesothelioma cells were directly stimulated to replicate or increase DNA synthesis by any of the asbestos exposure conditions tested. The potential selective effect of asbestos exposure was demonstrated by a differential sensitivity of normal mesothelial cells and mesothelioma cells to amosite: for example, up to 20-fold higher concentrations of amosite fibers were required to inhibit replication of mesothelioma cell lines than normal mesothelial cells. In addition, a significant resistance (4-fold) to amosite toxicity was observed for SV40 immortalized mesothelial cell lines that had previously been selected in vitro for resistance to asbestos. SV40 immortalized cells that have become tumorigenic after transfection with either Ha-ras or PDGF A-chain genes were not significantly more resistant to the cytotoxic effects of amosite than primary normal cells, and the primary cells were equally sensitive to amosite as mesothelial cells that were only immortalized by SV40. The sensitivity of normal mesothelial cells to asbestos does not appear to be simply a result of general fragility of the mesothelial cells, since similar levels of hydrogen peroxide and silica were cytotoxic for normal mesothelial cells and mesothelioma cell lines. Because mesothelioma cells have a greater resistance to asbestos cytotoxicity than normal mesothelial cells, we hypothesize that a differential resistance to cell killing by asbestos fibers in vivo may result in a selective expansion of an initiated or transformed cell population and thus contribute to the carcinogenesis process. Since tumorigenicity and asbestos resistance occur independently of one another in genetically altered mesothelial cell lines, genotypic and phenotypic alterations that lead to tumorigenic conversion may not be the same changes that provide resistance to cell killing by asbestos.

Adenocarcinoma of the ileostomy: the latent risk of cancer after colectomy for ulcerative colitis and familial polyposis.

A case of a primary adenocarcinoma of an ileostomy is reported along with 15 other cases collected from the literature. These rare tumors are seen on the average 24 years after colectomy with ileostomy and in all cases are associated with a past history of ulcerative colitis or familial polyposis. Most of the reported cases of these tumors have appeared in the literature within the past 5 years, suggesting that there is a rising incidence of this disease corresponding to completion of a biologic latency period that began when the Brooke ileostomy was introduced for ulcerative colitis in 1951. In our case a mucinous adenocarcinoma occurred at the ileostomy site 34 years after colectomy. Adjacent to the tumor was mucosa showing colonic metaplasia and focal dysplasia. Subsequent biopsy specimens of the revised stoma showed inflammatory lesions morphologically suggestive of inflammatory (pseudo) polyps. The clinical and morphologic features in this case suggest that there is transition from ileal mucosa to colonic mucosa to colonic dysplasia to adenocarcinoma. Annual evaluation of the ileostomy for colonic metaplasia, inflammatory lesions consistent with ulcerative colitis and dysplasia, is recommended. In the presence of dysplasia, stomal revision is advised. Wide local excision is advised for adenocarcinoma.

Suppression of tumorigenicity of a human lung carcinoma line by nontumorigenic bronchial epithelial cells in somatic cell hybrids.

Hybrid cell lines between HuT292-DM, a human lung carcinoma line resistant to 6-thioguanine and ouabain, and either normal human bronchial epithelial cells (NHBE) or an SV40 "immortalized" but nontumorigenic derivative thereof (BEAS-2B), have been isolated by double selection. Hybrids of NHBE and HuT292-DM cells senesced after 40-43 population doublings in culture. In contrast, hybrids of BEAS-2B and HuT292-DM showed no sign of a culture "crisis" and have an indefinite life span. HuT292-DM cells produced tumors in 100% of athymic nude mice with a mean latency of 27 days, whereas tumorigenicity was totally suppressed in 76% of the BEAS-2B x HuT292-DM hybrids, with a 2- to 3-fold increased tumor latency in the remaining 24% of these hybrids. While the hybrids are hypotriploid to hypotetraploid, the parental lines are hypodiploid. The growth of HuT292-DM cells is stimulated, whereas NHBE and BEAS-2B cells are inhibited by serum. The growth response of the BEAS-2B x HuT292-DM hybrids to serum is similar to that of HuT292-DM cells. Thus, tumorigenicity and culture longevity are dominantly controlled by the nontumorigenic parent (NHBE or BEAS-2B). On the other hand, serum responsiveness is more similar to that of the tumorigenic parent (HuT292-DM).

Mycobacterial disease associated with aplastic anaemia.

Haematological changes associated with infectious disease are relatively common but true aplastic anaemia secondary to infection is rare. We describe a patient wit disseminated Mycobacterium avium-intracellulare infection and who had a histologically proven remission of his aplastic anemia accompanying antimycobacterial therapy. We also review the literature on the haematological changes associated with mycobacterial infections and other infectious causes of aplastic anaemia.

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals.

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors.

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.