Biomedical subjects

C A Heckman

Publications and source records attributed to C A Heckman.

At least 19 recordsLinked to original sources

PubMed2006

The immunoglobulin heavy-chain gene 3' enhancers deregulate bcl-2 promoter usage in t(14;18) lymphoma cells.

In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.

Acetylation↗

PubMed2006

Oct transcription factors mediate t(14;18) lymphoma cell survival by directly regulating bcl-2 expression.

Oct-1 and Oct-2 are members of the POU homeodomain family of transcriptional regulators and are critical for normal embryonic development. Gene-targeting studies showed that Oct-1 and Oct-2 are largely dispensable for B-cell development and immunoglobulin production, although both Oct-2 and Bob-1 are required for a proper immune response and germinal center formation. In these studies, we investigated the role of Oct factors in B-cell lymphomas. Recent investigations have shown increased expression of Oct-2 and Bob-1 in lymphomas, and we observed greatly increased levels of Oct-2 in lymphoma cells with the t(14;18) translocation. Decreased expression of Oct-1, Oct-2, or Bob-1 by RNA interference resulted in apoptosis and down-regulation of bcl-2 expression. Furthermore, Oct-2 induced bcl-2 promoter activity and mediated this effect through three regions in the bcl-2 P2 promoter. Although these regions did not contain canonical octamer motifs, we observed the direct interaction of Oct-2 with all three sites both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. Moreover, by mutation analysis we found that the ability of Oct-2 to activate bcl-2 required C/EBP, Cdx, and TATA-binding sites. Oct-2, therefore, acts as a cell survival factor in t(14;18) lymphoma cells by directly activating the antiapoptotic gene bcl-2.

Apoptosis↗

PubMed2004

Novel p21-activated kinase-dependent protrusions characteristically formed at the edge of transformed cells.

During long-term culture, certain lines become neoplastic while accumulating changes in cell shape. Early and late cell populations have characteristic shape phenotypes that have been quantified by computerized assay. Phenotypes are determined from variables describing three-dimensional aspects of the subcellular distribution of mass. The features of cells can be recognized by use of latent factors, which are theoretical variables based on the covariance of the primary variables. Factor #7 represented a cell edge feature different from filopodia. We studied the morphological characteristics and morphogenesis of the feature. Brief exposure of cells from rat tracheal epithelium to phorbol 12-myristate 13-acetate (PMA) enhanced #7 values. The time to reach maximal #7 values was prolonged if PMA was administered with calcium ionophore or lysophosphatidic acid (LPA). Factor #7 was elevated during periods of ruffling suppression and stress fiber reorganization. Cells showing high #7 values were examined by scanning electron microscopy (SEM) and found to exhibit strap-shaped and cupola-shaped projections. Because RhoA regulates stress fiber formation, we sought to perturb #7 features by introducing dominant-acting negative and positive constructs of RhoA, RhoA-N19, and RhoA-V14. Neither affected #7 values. Although overexpression of the kinase inhibitory domain of p21-activated kinase 1 (PAK) had no effect on #7 values, they were affected by overexpression of a domain binding PAK-interacting guanine nucleotide exchange factor (PIX). Because a PAK-PIX complex is implicated in the remodeling of focal complexes (FCs) and recycling of PAK to the cytoplasm, the results implicate a component of FCs in the formation of #7 features. The data suggested that feature formation is driven by activated Cdc42-binding kinase (ACK) and Rac. Moreover, they suggested that the #7 protrusions are neurite-like structures and that their development involves FC regulation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2001

Molecular mechanisms of transcriptional control of bcl-2 and c-myc in follicular and transformed lymphoma.

A synergistic interaction of Bcl-2 and c-Myc plays a role in lymphomagenesis in mice and in some patients as well. Progression of follicular lymphoma to a more aggressive lymphoma is seen in the majority of patients, and approximately 10% of the transformed lymphomas have a translocation of c-myc in addition to the translocation of bcl-2 found in the original follicular lymphoma. We investigated whether transcriptional deregulation of bcl-2 and c-myc could be examined in primary lymphoma cells by in vivo footprinting and in vitro protein-DNA binding studies. A matched pair of follicular and transformed lymphoma samples was examined. The transformed lymphoma had acquired a translocation of c-myc into the immunoglobulin heavy chain locus. High levels of bcl-2 expression were observed in both the follicular and transformed lymphomas, whereas the expression of c-myc was low in the follicular lymphoma and increased in the transformed lymphoma. In vivo footprint analysis revealed that a CRE site and a Cdx site in the bcl-2 promoter were occupied on the translocated alleles but not on the normal alleles in both the follicular and transformed lymphomas. Two nuclear factor kappaB sites were occupied on the translocated c-myc allele in the transformed lymphoma. Gel shift analysis revealed that these proteins bound to their respective sites in the bcl-2 or c-myc promoter. There was no evidence that the presence of one of the translocations in the immunoglobulin heavy chain locus influenced the expression of the other translocated gene.

Alleles↗

PubMed2001

Negative regulation of bcl-2 expression by p53 in hematopoietic cells.

The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.

Apoptosis↗

PubMed2001

Mathematical modeling of marker influx and efflux in cells.

The tumor promoter, phorbol 12-myristate 13-acetate (PMA), affects the processing of fluid that enters a cell from the ambient medium. Previous work showed that marker accumulates to a higher level in PMA-treated than in untreated cells. Since PMA also affects the physical activity of the membrane and stimulates the normal process of taking up extracellular fluid, called endocytosis, it is important to learn whether the perturbations in fluid processing can be attributed entirely to a change in the cell's limiting membrane. To this end, a model for fluid uptake and processing was developed and applied to experiments in which a marker for extracellular fluid was added to cells. From previous work on marker accumulation, it was deduced that there were at least two functional compartments involved in fluid movement. Compartment I is a rapidly filling and rapidly recycling compartment. Compartment II is a slowly filling and emptying compartment. Three routes of vesicle traffic must be considered, one mediating influx from the ambient medium into compartment I, a second, efflux from compartment I to the medium, and a third efflux from compartment I into compartment II. Using earlier models for processing, workers found it difficult to estimate rates of movement through either of the latter routes, as well as the volume of compartment I. The difficulty arises from the fact that only one kinetic constant can be estimated directly from data, namely the instantaneous uptake rate. The remaining data depend on measuring the total mass of marker in the cells. Since the concentration of marker in the cell changes continuously, it is advantageous to employ differential equations to simulate the tracer movement. By applying the model to experimental values, we found estimates for all three rates of fluid movement and the volume of compartment I. It is thought that the model will enable us to determine whether apparent alterations in the time course of uptake arise solely from altered properties of the limiting membrane.

Animals↗

PubMed2000

A-Myb up-regulates Bcl-2 through a Cdx binding site in t(14;18) lymphoma cells.

In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.

Animals↗

PubMed2000

Enhancement of the transformed shape phenotype by microtubule inhibitors and reversal by an inhibitor combination.

Differences between transformed cells and their normal counterparts were defined by analyzing the cells' three-dimensional distribution of mass. Variables called factors, which explained the covariance of the real variables, were extracted from the data. We found that factors #4 (sharp, tapering projections) and #12 (rounding up), corresponded to G-protein functions. Then, the signature-type mass distribution of transformed cells was defined by factor values. Agents that caused signature-type mimicry could quantitatively shift factor values, for example, those affecting endocytic processing disproportionately reduced values of #4. Signature-type reversal was also observed and may be valuable in predicting the efficacy of chemotherapeutic agents.

Animals↗

PubMed1999

Describing shape dynamics in transformed cells through latent factors.

Previous studies attributed the characteristic shape changes found in cancer cells, in part, to aberrant vesicle traffic. Typically, transformed cells also rounded up. These phenomena were further investigated by measuring the shape features of cells from established lines, which represented both normal and oncogenic stages of transformation. Although conventional pattern recognition methods, applied to a combined data set from these lines, failed to reveal any new, recognizable features beyond those already known, factors did describe such features. Factors are hypothetical variables that contribute to the variance of two or more measurable variables. One factor for the cell edge, 5, was known from previous studies on correlations among the variables. Several other factors at the same level identified crucial features. Factor 4 reflected the frequency of microspikes; another factor described a knob-like structure (7). A third, factor 16, indexed the variability in projection size. Factors of the upper cell, 1 micrometer or more above the substratum, namely, 1, 2, 8, 11, 13, and 19, also described transformation-related changes. Comparing lines that modeled the development of bronchogenic carcinoma, we found a tendency for 2 (surface smoothing), 4, and 12 (rounding-up) to be changed irreversibly. Thus, factors overcame the problem of relating mathematical shape phenotypes, previously obtained based on single variables, to cell features.

Animals↗

PubMed1999

Soybean isoflavones trigger a calcium influx in Phytophthora sojae.

Both the motile zoospores and the hyphal germ tubes of Phytophthora sojae respond chemotropically to the soybean isoflavones daidzein and genistein. The role of Ca(2+) in the cellular response to these host signals was investigated by using X-ray microanalysis of cells to monitor net changes in cellular levels of Ca(2+) and by quantifying the effects of exogenous Ca(2+) and daidzein on the developmental fate of encysted zoospores. Confirmation that isoflavones trigger a net influx of Ca(2+) into the cell was demonstrated by X-ray microanalysis of individual encysted zoospores. Zoospores exposed to 10 mM Ca(2+) and 1 microM daidzein at the time of encystment formed cysts that contained more Ca(2+) than zoospores exposed to Ca(2+) alone. The magnitude of internal Ca(2+) stores appears to be a determining factor affecting the developmental fate of P. sojae cysts.

Calcium↗

PubMed1998

The sevenfold way of PKC regulation.

Protein kinase C (PKC) is a family of enzymes that are physiologically activated by 1,2-diacylglycerol (DAG) and other lipids. To date, 11 different isozymes, alpha, betaI, betaII, gamma, delta, epsilon, nu, lambda(iota), mu, theta and zeta, have been identified. On the basis of their structure and activators, they can be divided into three groups, two of which are activated by DAG or its surrogate, phorbol 12-myristate 13-acetate (PMA). PKC isozymes are remarkably different in number and prevalence in different cell lines and tissues. When activated, the isozymes bind to membrane phospholipids or to receptors that are located in and anchor the enzymes in a subcellular compartment. Some PKCs may also be activated in their soluble form. These enzymes phosphorylate serine and threonine residues on protein substrates, perhaps the best known of which are the myristoylated, alanine-rich C kinase substrate and nuclear lamins A, B and C. The enzymes clearly play a role in signal transduction, and, because of the importance of PMA as a tumor promoter, they are thought to affect some aspect of cell cycling. How PKC takes part in the regulation of cell transformation, growth, differentiation, ruffling, vesicle trafficking and gene expression, however, is largely unknown.

Animals↗

PubMed1996

Persistent effects of phorbol 12-myristate 13-acetate: possible implication of vesicle traffic.

Relative to their normal counterparts, transformed epithelial cells have a distinctive and quantifiable three-dimensional shape. Biophysical and mathematical methods are used to distinguish these extremes in cells from two lines, cultured from rat liver and tracheal epithelium, respectively. Cells adopted a more transformed-looking configuration transiently when exposed to phorbol 12-myristate 13-acetate (PMA) (Plummer and Heckman, [1990] Exp. Cell Res., 188:66-74). The purpose of the present work was to dissect the physiological processes involved in the shape change. Ruffling activity, known to be PMA-stimulated in other cells, was investigated. Although the ruffles appeared less robust than normal, PMA stimulated ruffling activity over a 5 h period. The number of sites where ruffling was initiated declined by 5 h, however, and suppression was seen by 10 h. Cells from both lines adopted the transformed shape configuration when exposed for 2 h to monensin. When the subset of shape features changed by this treatment was compared with those originally changed during transformation, it was found that monensin-treated cells mimicked the features of transformed cells. Its effect on ruffling was, however, unlike PMA's. Thus, the phenotype was unlikely to arise from ruffling itself but might be a process driven by ruffling. Chloroquine also stimulated cells to assume characteristics of transformed cells. Since both it and monensin could interfere with endosomes and with the processing of endocytosed contents, this was a likely site of action. Experiments were done to determine whether PMA also affected the processing of extracellular fluids. When the accumulation of horseradish peroxidase (HRP) was measured, the rate was found to be higher in PMA-treated cells from 5 min, the earliest time assayed, onward. The results suggest that the transformed type of cell in these cell lines showed a constitutive dilation and/or reorganization of some portion of the endosomal pathway.

Animals↗

PubMed1995

Branchpoint and polypyrimidine tract mutations mediating the loss and partial recovery of the Moloney murine sarcoma virus MuSVts110 thermosensitive splicing phenotype.

Balanced splicing of retroviral RNAs is mediated by weak signals at the 3' splice site (ss) acting in concert with other cis elements. Moloney murine sarcoma virus MuSVts110 shows a similar balance between unspliced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N-nitroso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicing was virtually complete at all temperatures and have investigated the molecular basis of this reversion on the assumption that the findings would reveal cis-acting elements controlling MuSVts110 splicing thermosensitivity. In a representative revertant (NMU-20), we found that complete splicing was conferred by a G-to-A substitution generating a consensus branchpoint (BP) signal (-CCCUGGC- to -CCCUGAC- [termed G(-25)A]) at -25 relative to the 3' ss. Weakening this BP to -CCCGAC- [G(-25)A,U(-27)C] moderately reduced splicing at the permissive temperature and sharply inhibited splicing at the originally nonpermissive temperature, arguing that MuSVts110 splicing thermosensitivity depends on a suboptimal BP-U2 small nuclear RNA interaction. This conclusion was supported by results indicating that lengthening the short MuSVts110 polypyrimidine tract and altering its uridine content doubled splicing efficiency at permissive temperatures and nearly abrogated splicing thermosensitivity. In vitro splicing experiments showed that MuSVts110 G(-25)A RNA intermediates were far more efficiently ligated than RNAs carrying the wild-type BP, the G(-25)A,U (-27)C BP, or the extended polypyrimidine tract. The efficiency of ligation in vitro roughly paralleled splicing efficiency in vivo [G(-25)A BP > extended polypyrimidine tract > G(-25)A,U(-27)C BP > wild-type BP]. These results suggest that MuSVts110 RNA splicing is balanced by cis elements similar to those operating in other retroviruses and, in addition, that its splicing thermosensitivity is a response to the presence of multiple suboptimal splicing signals.

Amino Acid Sequence↗

PubMed1993

Ruffling and locomotion: role in cell resistance to growth factor-induced proliferation.

It has long been known that the growth rate of cells in vitro can be retarded by providing substrates of restricted area. Such experiments were performed with adhesive islets, made by depositing metals onto agarose layers through templates of various sizes. Since normal cells are unable to adhere to agarose, they become confined to the metallic surface. Using such haptotactic islets, we have studied the role of membrane ruffling and cell locomotion in the resistance of AG1523 human fibroblasts to growth factor-induced mitogenesis. Cells plated on small substrates, i.e., 2,150 microns 2 in area, initially showed vigorous ruffling, which was suppressed by 8 h after plating but had resumed again by 12 h. In contrast, cells on larger-size islets showed a rapid decline and stabilization of ruffling activity. When the growth rate was measured for single cells cultured on haptotactic islets, it was found to increase linearly from areas of 4,280 microns 2 up to 425,000 microns 2. Since the area needed to saturate the growth response was approximately 50-fold larger than the area occupied by a single cell, the growth inhibition was attributed in part to an interference with locomotion. The implication that locomotion provided positive input into growth control mechanisms was subjected to a direct test by evaluating the effect of nine polypeptide growth factors on the motility of serum-starved cells. All except TGF-beta 1 stimulated movement. Finally, the mitogenic effect of growth factors was measured by [3H]thymidine incorporation and found to be proportional to motile activities, as quantitatively assayed. We conclude that locomotion suppression is a factor in AG1523 cell resistance to growth factor-induced mitogenesis.

Cell Division↗

PubMed1990

Geometrical constraints on the shape of cultured cells.

Previous studies on cultured cells revealed that the values of certain shape descriptors were highly correlated despite the fact that there was little mathematical interdependence (Heckman, CA: In Advances in Cell Culture, Vol. 4, K. Maramorosch, ed, pp 85-156, 1985). The independence of such descriptors was tested by calculating the values of correlations among descriptors for a data set consisting of macroscopic objects. The descriptors were unlinked for this data set, confirming that high correlations reflected specific characteristics of cultured cells. These characteristics were identified by constructing model figures incorporating the geometrical features postulated to be responsible for each correlation. Because the position of the ellipse of concentration depended upon the form in which mass was displaced in figures, the fraction of the figure falling within the area of the ellipse (FINE) and the ratio of the ellipse area to the figure's area (ARAT) became decorrelated from each other in figures with massive projections. Further, correlations between ARAT or FINE and variables measuring cavities and inflections of the perimeter showed a complex dependence on the size, shape, and number of invaginations. Descriptors could become decorrelated due to features present in only a minority of cells of a population. Finally, cell populations that differed with respect to physiological characteristics showed no differences in the value of ARAT:FINE correlation. However, correlations between ARAT and the coefficient of variation of centroid-to-perimeter distances were altered. This suggested that correlation coefficients may be themselves powerful descriptors of the shape characteristics of cell populations.

Animals↗

PubMed1990

Transient expression of the transformed phenotype stimulated by 12-O-tetradecanoyl phorbol-13-acetate.

The objective of the present work was to determine whether a short-term perturbation of cells by 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment caused shape changes identical to those found in oncogenic transformation. A cell line derived from the rat respiratory tract epithelium, 1000 W, was used, in which shape changes had been identified previously as the cells underwent transformation during long-term growth in vitro. These changes corresponded to a steeper rise of the cell from the substrate and a smoothing of the surface contours throughout the periphery of the cell. The phenotype was measured by maximum likelihood estimation, based on the values of several geometrical shape descriptors. With continuous TPA treatment, the cells adopted a transformed phenotype by 2 h. The effect was maximal by 5 h but began to decline by 10 h. Shape change in the opposite direction was stimulated by treatment with the protein kinase C inhibitor staurosporine, and its effects were counteracted if the cells were simultaneously exposed to TPA. No appreciable metabolism of [20-3H]TPA occurred until 24 h after treatment. Enumerating the shape descriptors whose values composed the transformed phenotype indicated that the TPA-stimulated changes were qualitatively similar to those accompanying oncogenic transformation. The subsequent alterations, however, involved few of the variables that composed the transformed phenotype and therefore did not represent a true reversal of the change. Changes observed up to 5 h were not dependent on new RNA synthesis but required continued protein synthesis.

Animals↗

PubMed1987

Characteristic shape and surface changes in epithelial transformation.

Previous work has suggested that adhesive or cytoskeletal structures alter during epithelial transformation. The purpose of the present studies was to see whether such reorganizations were accompanied by consistent changes in the biophysical characteristics of cells. We used in vitro cell lines from rat liver and respiratory tract epithelium that became tumorigenic over a period of 11-15 months. For populations sampled at several time points before and after this transition, the shape of individual cells was analysed by calculating the values of many geometrical descriptors. The data were collected from cells imaged by reflected light interference. Interference contours on the cells were digitized, and the descriptor values calculated by computer program. Cell lines derived from both liver (IAR 20PC1) and respiratory epithelium 1,000 W) lines showed progressive changes in the values of certain descriptors. The time-dependent differences among groups were statistically significant at a level of p less than 0.0001. By determining which descriptors underwent large changes, we found that some structural revisions occurred in common in the two types of cells. Cells from later passages of the lines rose from the substrate at a steeper angle, as determined by measuring the fraction of the area of the first interference contour that was occupied by successive contours. In addition, the projections on the perimeter became broader, and the perimeter became less complex. This kind of 'smoothing' of the cell perimeter took place in several contours, indicating that it was a surface change. Surface changes of this sort have not been noted previously in transformation. The results suggest that the structure of cells is altered in a consistent way during transformation, as detected by measuring of shape and surface configuration.

Animals↗

PubMed1985

Ultrastructure and distribution of intracellular spicules in rat lung following chronic tobacco smoke exposure.

Factors that contribute to the induction of lung tumors in F344 rats by chronic tobacco smoke exposure were studied. Previous studies had shown that tobacco smoke exposures of 1-2 years duration induced only one major type of lesion involving the respiratory airways, i.e., fibrotic and cellular enlargement of peribronchiolar alveolar septa. The airway epithelium in these areas was metaplastic and in some of the lesions, the airway lining epithelium advanced out onto the surfaces of adjacent alveoli. Epithelial cells in these lesions frequently contained elongated cytoplasmic inclusions which were oriented with their long dimensions roughly in the same plane as the long axis of the cell. Macrophages contained similar but larger inclusions. Because the composition of the inclusions could be indicative of their origin, we subjected samples of treated and control lung tissues concurrently to transmission electron microscopy and energy-dispersive X-ray fluorescence spectrometry. Spectra from inclusions of macrophages indicated the presence of the elements sulfur, phosphorus, aluminum, silicon, and iron. Spectra from type II cells, however, which did not contain inclusions, showed a different elemental composition. The results suggested that spicules were present in epithelial cells throughout the airways. Minor lesions corresponding to "microinvasion" of epithelium into the lamina propria and of capillaries into the epithelial layer were also found in the trachea.

Animals↗