Risk of basal cell carcinoma in a randomized clinical trial of aspirin and folic acid for the prevention of colorectal adenomas.

BACKGROUND: Aspirin may reduce the risk of several types of cancer. OBJECTIVES: To evaluate if folic acid is associated with risk of basal cell carcinoma (BCC). METHODS: BCC incidence was evaluated in a randomized, double-blind, placebo-controlled clinical trial of aspirin (81 mg daily or 325 mg daily for ~3 years) and/or folic acid (1 mg daily for ~6 years) for the prevention of colorectal adenomas among 1121 participants with a previous adenoma. BCC was confirmed by blinded review of pathology reports. RESULTS: One hundred and four of 958 non-Hispanic white participants were diagnosed with BCC over a median follow-up of 13·5 years. Cumulative incidence of BCC was 12% [95% confidence interval (CI) 7-17] for placebo, 16% (95% CI 11-21) for 81 mg aspirin daily and 15% (95% CI 10-20) for 325 mg aspirin daily [hazard ratio (HR) for any aspirin 1·45 (95% CI 0·93-2·26); HR for 81 mg daily 1·57 (95% CI 0·96-2·56); HR for 325 mg daily 1·33 (95% CI 0·80-2·20)]. BCC risk was higher with aspirin use in those without previous skin cancer but lower with aspirin use in those with previous skin cancer (Pinteraction = 0·02 for 81 mg aspirin daily; Pinteraction = 0·03 for 325 mg aspirin daily). Folic acid supplementation was unrelated to BCC incidence (HR 0·85; 95% CI 0·57-1·27). CONCLUSIONS: Neither aspirin nor folic acid treatment had a statistically significant effect on risk of BCC. Subgroup analysis suggested that chemopreventive effects of nonsteroidal anti-inflammatory drugs may be specific to those at high risk for BCC.

Expression of mRNAs for the alpha 1 subunit of voltage-gated calcium channels in human osteoblast-like cell lines and in normal human osteoblasts.

The activation of osteoblast calcium channels by many bone regulatory factors suggests an important role for intracellular calcium signaling in the control of bone remodeling. At least six different genes for the alpha 1 subunit of voltage-gated calcium channels have been cloned including L-type (alpha 1S, alpha 1C, and alpha 1D) and non-L-type (alpha 1A, alpha 1B, and alpha 1E) isoforms. The goal of the present study was to identify which of these calcium channel isoforms are transcribed in human osteoblast-like cell lines (hFOB, MG-63, SAOS-2, TE-85, G-292) and in cultures of normal human osteoblasts. Reverse transcriptase-PCR was used to amplify sequences corresponding to each of the alpha 1 subunits using isoform specific primers. The products of the PCR reaction were cloned and sequenced to verify their identify and used to probe southern blots of the PCR reactions. The results indicate that among the different types of osteoblast-like cells examined, two calcium channel isoforms were always expressed (alpha 1C and alpha 1A), three isoforms were variably expressed (alpha 1S, alpha 1D and alpha 1B), and one isoform was not expressed in any of the osteoblast-like cells (alpha 1E) but was easily detected in human brain controls. Our results indicate that mRNAs for multiple calcium channel alpha 1 subunits are expressed in human osteoblasts, including both L-type and non-L-type isoforms. In addition, significant heterogeneity exists between the different osteoblast cell models examined in the type and mRNA abundance of the different calcium channel isoforms.

Regulation of renal parathyroid hormone receptor expression by 1, 25-dihydroxyvitamin D3 and retinoic acid.

The renal distal convoluted tubule (DCT) is the major site of parathyroid hormone (PTH) and 1alpha,25-dihydroxyvitamin D3 [1, 25(OH)2D3]-regulated calcium absorption. 1,25(OH)2D3 augments PTH-stimulated calcium transport by DCT cells, while having no effect of its own. 1,25(OH)2D3 mediates its effects on gene expression by binding to a nuclear vitamin-D receptor (VDR), which then associates with the retinoid-X receptor (RXR) as a heterodimer. We studied the effects of 1,25(OH)2D3, 9-cis- and all-trans-retinoic acid on PTH/PTHrP receptor expression. mRNAs for the PTH/PTHrP, VDR, and RXR receptors were detected in immortalized DCT cells by reverse transcriptase-polymerase chain reaction. Changes in PTH/PTHrP receptor mRNA expression were quantified by slot blot hybridization. 1,25(OH)2D3 maximally increased PTH/PTHrP receptor mRNA levels by 70%. The stimulation was specific since 1,25(OH)2D3 treatment had no effect on the expression of adrenergic receptor or Na+/H+ exchanger mRNA levels. Likewise, the inactive form, 25(OH)2D3 had no effect on PTH/PTHrP receptor mRNA expression. In combination with the putative RXR ligand, 9-cis-retinoic acid, 1,25(OH)2D3 increased PTH/PTHrP receptor mRNA levels 4-fold. 9-cis-Retinoic acid had no effect of its own on steady-state PTH/PTHrP receptor mRNA expression. The putative ligand for the retinoic acid receptor, all-trans-retinoic acid, increased PTH/PTHrP receptor mRNA expression alone and in combination with 1,25(OH)2D3. 9-cis-Retinoic acid alone, and in combination with 1,25(OH)2D3, also increased specific PTH/PTHrP receptor binding to plasma membranes isolated from DCT cells. These results indicate that 1,25(OH)2D3 upregulated PTH/PTHrP receptor expression at both mRNA and protein levels in a manner consistent with VDR/RXR heterodimers transactivating the PTH/PTHrP receptor gene by binding a vitamin D response element in the PTH/PTHrP gene.

Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in transporting epithelial cells.

Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

Expression of the sodium-chloride cotransporter in osteoblast-like cells: effect of thiazide diuretics.

The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

Multiple calcium channel transcripts in rat osteosarcoma cells: selective activation of alpha 1D isoform by parathyroid hormone.

Osteoblasts express calcium channels that are thought to be involved in the transduction of extracellular signals regulating bone metabolism. The molecular identity of the pore-forming subunit (alpha 1) of L-type calcium channel(s) was determined in rat osteosarcoma UMR-106 cells, which express an osteoblast phenotype. A homology-based reverse transcriptase-polymerase chain reaction cloning strategy was employed that used primers spanning the fourth domain. Three types of cDNAs were isolated, corresponding to the alpha 1S (skeletal), alpha 1C (cardiac), and alpha 1D (neuroendocrine) isoforms. In the transmembrane segment IVS3 and the extracellular loop formed by the IVS3-S4 linker, a single pattern of mRNA splicing was found that occurs in all three types of calcium channel transcripts. Northern blot analysis revealed an 8.6-kb mRNA that hybridized to the alpha 1C probe and 4.8- and 11.7-kb mRNAs that hybridized to the alpha 1S and alpha 1D probes. Antisense oligonucleotides directed to the calcium channel alpha 1D transcript, but not those directed to alpha 1S or alpha 1C transcripts, inhibited the rise of intracellular calcium induced by parathyroid hormone. However, alpha 1D antisense oligonucleotides had no effect on the accumulation of cAMP induced by parathyroid hormone. When L-type calcium channels were activated with Bay K 8644, antisense oligonucleotides to each of the three isoforms partially inhibited the rise of intracellular calcium. The present results provide evidence for the expression of three distinct calcium channel alpha 1-subunit isoforms in an osteoblast-like cell line. We conclude that the alpha 1D isoform is selectively activated by parathyroid hormone.

Expression and antibody inhibition of P-type calcium channels in human small-cell lung carcinoma cells.

P-type channels, a recently described form of voltage-gated calcium channels, are found in many central and peripheral neurons. In the present study, a partial cDNA clone sharing extensive nucleotide identity with a putative P-type voltage-gated calcium channel alpha 1 subunit was isolated from a small-cell lung carcinoma (SCLC) cell line. Anti-peptide antibodies generated to a unique acidic stretch in the IVS5-S6 linker region of the putative SCLC P-type channel reacted specifically with a SCLC fusion protein produced in bacteria and with a cell surface molecule in SCLC cells. Calcium currents in SCLC cells, measured by whole-cell patch clamp, were inhibited by these antibodies and by the P-type channel-specific toxin omega-agatoxin IVA. The inhibitory effects of the antibody and the toxin were not additive, consistent with their proposed action on the same type of channel. These results provide evidence for the expression of P-type calcium channels by SCLC cells. The expression of neuron-related molecules by these cells is of particular interest because small-cell lung carcinoma is frequently associated with paraneoplastic disorders affecting the nervous system.

Introduction of antisense oligonucleotides into cells by permeabilization with streptolysin O.

Cellular uptake of antisense oligonucleotides is critical to their ability to inhibit gene expression. In the present study, phosphodiester oligodeoxynucleotides were introduced into cells during brief permeabilization with the pore-forming agent streptolysin O. The extent of antisense inhibition was dependent on the concentration of oligonucleotide present during permeabilization. In addition, the level of antisense inhibition was time-dependent; it reached a maximum at 18 h and subsequently diminished to control levels over the next 48 h. These results demonstrate the effectiveness of streptolysin O permeabilization as a means for simple and rapid introduction of oligonucleotides into eukaryotic cells.

Assembly of fibronectin into extracellular matrix.

The results summarized above suggest that assembly of fibronectin is a fundamental biological process and that knowledge of the process of assembly may reveal new ways by which cells interact with extracellular molecules. Deposition of a fibronectin matrix seems to be regulated as tightly as synthesis of fibronectin or expression of adhesion receptors for fibronectin and is influenced profoundly by two products of blood coagulation--TGF-beta released from platelets and factor XIII activated by thrombin. Fibronectin assembly may be important in all sorts of physiological and pathophysiological processes. Cell A--for instance, a stromal cell--can influence the behavior of cell B--for instance, a lymphocyte--by assembling fibronectin made by cell C--for instance, a hepatocyte. We hope that the testable models of assembly presented in this paper will lead to new understanding of the process of assembly and suggest new modalities for treatment of diseases that result in fibrosis, damaged tissues, and neoplastic growth.

Binding and degradation of blood coagulation factor XIII by cultured fibroblasts.

The interaction of Factor XIII with cultured fibroblasts was examined using 125I-labeled protein and immunofluorescence. Platelet or plasma Factor XIII bound to confluent cell layers. Binding reached an apparent steady state after 8 h. Activation with thrombin increased the binding of both the platelet and plasma forms of the enzyme. After a 1-2 h lag, a chloroquine-inhibitable increase in trichloroacetic acid-soluble radioactivity was detected in the medium. Gel electrophoresis in sodium dodecyl sulfate indicated that approximately 16-fold more a subunit (catalytic) of 125I-plasma Factor XIII bound to the cell layer than b subunit (carrier) and that some large complexes containing Factor XIII were formed with the cell layer. Factor XIII binding increased linearly with concentrations of Factor XIII up to 230 micrograms/ml, whereas a component of the degradation of Factor XIII was saturable at about 20 micrograms/ml. Factor XIII associated with cell layers was catalytically active since it could cross-link fibronectin. By immunofluorescence the a subunit of Factor XIII was localized to fibronectin-containing extracellular fibrils and, in the presence of chloroquine, to intracellular granules. These results indicate that the a subunit of Factor XIII binds to the fibroblast extracellular matrix and matrix assembly sites, where it remains active, and to a putative cell-surface receptor which mediates its internalization and degradation.

Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments.

Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites.

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.