Marlene Absher

Tamoxifen induces TGF‐β1 activity and apoptosis of human MCF‐7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF‐7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF‐7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM‐2 and TGF‐β1 mRNAs in MCF‐7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen‐induced pS2 gene was strongly suppressed. The biological activity of TGF‐β was increased at least fourfold in the media from MCF‐7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF‐7 cells and it may be mediated by the secretion of active TGF‐β.

Comparative Clearance of Quartz and Cristobalite from the Lung

The two silicon dioxide polymorphs, quartz and cristobalite, are known to have different toxicities. The clearance kinetics and biological response of two sources of quartz and one source of pure cristobalite were compared. Models were also developed to show the accumulation of cristobalite in the lungs of Fischer 344 rats as the result of short-term exposures at three different concentrations. The amount of cristobalite cleared from the lung was considerably less than that of the two quartz materials, with little or no clearance after the initial 30 days post exposure. As an indicator of the cellular biological response to the aerosols, total and differential cell counts were measured on bronchoalveolar lavage specimens. Cristobalite showed an early and sustained response with an elevated macrophage, neutrophil, and lymphocyte count through 180 days post exposure. The two quartz materials were not identical in their biological behavior even though they had identical crystal structure and similar trace element analysis. One quartz sample (MIN5) showed an increase in cell response (macrophage, neutrophils, and lymphocytes) approximately 30% that of cristobalite, whereas the other quartz material was not significantly different from control values. In addition, lung hydroxyproline content was greater in the cristobalite-exposed animals.

Characterization of vascular smooth muscle cell phenotype in long-term culture

SummaryStudies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavoir, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concommitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression.

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express α- and γ-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Glucocorticoid-induced down regulation of transforming growth factor-β1 in adult rat lung fibroblasts

Transforming growth factor-β1 mRNA and transforming growth factor β activity are decreased with exposure of normal adult rat lung fibroblasts to dexamethasone. Dexamethasone caused a decrease in transforming growth factor-β1 mRNA within 2 hours, which was sustained at least over a 24-hour period. The decrease in transforming growth factor-β1 mRNA was dose related. Dexamethasone treatment of rat lung fibroblasts also resulted in a decrease of transforming growth factor β activity as determined by the mink lung cell growth inhibition assay. These data indicate that glucocorticoids may regulate collagen synthesis at least in part through the mediation of transforming growth factor-β1 in rat lung fibroblasts.

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establishment of a monolayer from a low-density seed (7.5 X 10(4) cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 x 10(6) cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 x 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture 1, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture 1). Interdivision times (IDT) were longer and relatively constant in culture 1 until near confluence; none were less than 10 h, whereas in 2, 24% of the IDTs were less than or equal to 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.

What is the relationship between hemolytic potential and fibrogenicity of mineral dusts

The hemolytic reaction to a dust is often used as a potential indicator of fibrogenicity of silicon dioxide polymorphs. However, occasionally the hemolytic response may not correlate with the observed fibrotic response in vivo. For example, amorphous silicas are very hemolytic but have little or no fibrogenic activity. In our study, heat treatment was used to alter alpha-cristobalite, a known fibrogenic dust, to a more hydrophobic surface. Comparisons were made between heated and unheated alpha-cristobalite for hemolytic activity in vitro and for lung response in vivo. Heat treatment resulted in decreased hemolytic response, but no change in the fibrotic response occurred in vivo. In addition, the heat treatment resulted in increased initial dust accumulation, reduced short-term clearance, and enhanced long-term clearance in vivo. Increased inflammatory cell recruitment was also observed in lungs of animals exposed to alpha-cristobalite. Thus, whereas heat-induced surface changes in alpha-cristobalite markedly altered the hemolytic activity of the particles, no changes were observed in the fibrotic response.

Altered rates of collagen synthesis in in vitro aged human lung fibroblasts

SummaryAbsolute rates of protein and collagen synthesis based on prolyl-tRNA as the precursor were determined in two age groups of IMR-90 human lung fibroblasts. Compared with midrange fibroblasts [population doubling level (PDL)=20 to 30] aged fibroblasts (PDL>40) were larger in siz in terms of protein and RNA per cell, generally proliferated more slowly, exhibited different steady state [3H]proline transfer RNA (tRNA) precursor pool specific radioactivities, synthesized collagen at a substantially lower rate, and exhibited a reduction in the percent commitment to collagen synthesis. Total protein synthetic rates were reduced slightly in aged versus midrange fibroblasts but the difference was not statistically significant. Proliferative capacity (PDL/wk) correlated better with these changes than cumulative PDL. Cell size (protein/cell) was the variable that had the highest correlation with the reduction in collagen synthesis observed in human lung fibroblasts. Thus, an important differentiated function of human lung fibroblasts, collagen synthesis, is greatly diminished in vitro in large, slowly dividing fibroblasts.

Increases in endogenous antioxidant enzymes during asbestos inhalation in rats

Although the pathogenesis of asbestos-induced pulmonary damage is still not completely understood, an important role has been attributed to active oxygen species. In the present paper we present results of a study investigating the effect of crocidolite asbestos inhalation on different lung antioxidant enzymes in rats. During the development of pulmonary fibrosis induced by crocidolite asbestos, lung superoxide dismutase, catalase and selenium-dependent glutathione peroxidase activities increased, indicating an adaptive response to increased pulmonary oxidant stress. However, this adaptive response obviously is not sufficient to protect the lung from asbestos-induced pulmonary damage. Considering the role of active oxygen species in both the fibrotic process and tumor promotion, it is hypothesized that antioxidants may also protect the lung from chronic asbestos-induced pulmonary damage such as bronchogenic carcinoma.

Intrathoracic Distribution and Transport of Aerosolized Silica in the Rat

Short-term exposure of rats to aerosols of the silicon dioxide, cristobalite, leads to pulmonary inflammation persisting several months. Clearance of particles occurs during the first two weeks after cessation of exposure, after which there is little additional clearance in the whole lung. In the present studies, quantitation of silica in lung compartments at selected times following exposure indicated movement of particles between the alveolar space and the lung tissue per se, with increased alveolar silica content associated with decreased silica content in the tissue compartment. Further, changes in the silica content in the alveolar compartment were generally associated with fluctuations in the alveolar macrophage population. Silica accumulated linearly in the mediastinal lymph nodes and thymus for several months after cessation of exposure, while negligible amounts were found in kidney, spleen, liver, and blood. A compartmental model was used to describe the distribution and translocation kinetics of the inhaled silica in the lung and extrapulmonary tissues.