Serum-deficient Medium Research Articles

Abstract B039: Influence of tumor-associated fibroblasts and their exosomes in the development and progression of prostate cancer

Abstract The unique tumor microenvironment (TME) of the prostate and in particular fibroblasts are known to play an essential role in the growth and progression of prostate cancer (PCa). Earlier studies from our group have shown that co-injection of patient-derived prostate cancer-associated fibroblasts (CAFs) and non-cancer associated fibroblasts (NCAFs) with tumor cells stimulated tumor growth in xenograft models compared to the injection of tumor cells alone. Our present study aims to investigate the effect of patient-derived prostate CAFs and NCAFs on the functional aspects of tumor cells in-vitro and their role in epigenetic regulation. Three prostate CAFs and NCAFs pairs were isolated from tissues of patients who had undergone radical prostatectomy and cultivated in-vitro. We then examined the influence of these fibroblast pairs on the viability, proliferation and migration of prostate cancer cell lines LNCaP and LNCaP C4-2. Cells were co-cultured in a transwell system with tumor cells in the lower chamber and fibroblasts in the upper chamber for 2 and 4 days and the viability of the tumor cells was measured using WST-1 assay and proliferation using BrdU colorimetric assay. For migration assay, the tumor cells were pretreated with serum-deficient medium and were co-cultured in a transwell system as mentioned above for 4 days. The tumor cells were collected after co-culture and the migration was measured using the transwell migration system as well as Xcelligence real-time assays in parallel. Further, extracellular vesicles (EV) were enriched from fibroblasts by ultracentrifugation. The expression of miRNAs isolated from cells and EVs was studied by microarray analysis and RT-qPCR. Patient-derived fibroblasts significantly increased the viability and proliferation of the tumor cells compared to the tumor cells alone. The influence of NCAFs on both viability and proliferation was more pronounced compared to CAFs on the LNCaP cells. However, the influence of the fibroblasts was vice versa on the LNCaP-C4-2 cells. The LNCaP cells exhibited no migration whereas the LNCaP C4-2 cells exhibited slight migration. The effect of different fibroblast pairs on the migration of LNCaP C4-2 cells varied among the pairs. 103 miRNAs and 6 miRNAs were found to be differentially expressed between CAFs and NCAFs and between their EVs, respectively. miR-10b-5p and miR-210-3p were confirmed by RT-qPCR to be upregulated in the CAFs. Our in-vitro data confirmed the in-vivo results. Fibroblasts modulate viability, proliferation and migration of PCa. However, the effects are heterogeneous due to patient and cell line-specific characteristics. Further studies will focus on the possible effects of fibroblasts on apoptosis in correlation to viability and invasion. CAFs and their EVs are characterized by specific miRNA expression supporting an epigenetic regulation of fibroblast characteristics and functions. Therefore, we will further investigate the mechanism of tumor progression induced by fibroblasts and their EVs via miRNA regulation. Citation Format: Aishwarya Tagat, Hiresh Ayoubian, Angela Zaccagnino, Michael Stöckle, Johannes Linxweiler, Kerstin Junker. Influence of tumor-associated fibroblasts and their exosomes in the development and progression of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B039.

Peroxisome proliferator-activated receptor gamma ligand-mediated apoptosis of hepatocellular carcinoma cells depends upon modulation of PI3Kinase pathway independent of Akt

BackgroundLigands of Peroxisome proliferator-activated receptor gamma (PPARγ) can inhibit growth and promote apoptosis in various cancer cells, and thus have the potential to be utilized as anticancer drugs. This potential however, has been seriously challenged by observations that they can lead to tumor promotion in some cancer models, possibly due to activation of different signaling mechanisms in various tumor environments. Elucidation of the specific signaling events that modulate PPARγ ligand-mediated events is thus critical to increase their efficacy. The studies described here were designed to elucidate the signaling pathway(s) that modulate the apoptotic potential of Troglitazone (TRG), an artificial PPARγ ligand in hepatocellular carcinoma (HCC) cells.ResultsOur results indicate that the apoptotic potential of TRG was regulated by the presence or absence of serum in the media. When added in serum-containing media, TRG inhibited proliferation and cyclin D1 expression, but was unable to induce any apoptosis. However, TRG's apoptotic potential was induced significantly when added in serum deficient media, as indicated by increased PARP and Caspase-3 cleavage and results from apoptosis assay. Furthermore, TRG-induced apoptosis in serum deficient media was associated with a dramatic reduction in PI3Kinase downstream target AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation. On the contrary, there was an increase of PI3K-induced AktSer473 and FoxO1Thr24/FoxO3aThr32 phosphorylation involving Pak, when TRG was added in serum-containing media. Pharmacological inhibition of PI3Kinase pathway with LY294002 inhibited Aktser473 phosphorylation and sensitized cells towards apoptosis in the presence of serum, indicating the involvement of PI3K in apoptosis resistance. Interestingly, pharmacological inhibition or siRNA-mediated knockdown of Akt or inhibition of Pak was unable to sensitize cells towards TRG-induced apoptosis in the presence of serum. Similarly, TRG was unable to induce apoptosis in the Akt1-KO, Akt1&2-KO MEFs in serum-containing media.ConclusionThese studies indicate that TRG-induced apoptosis is modulated by PI3K pathway in a novel Akt-independent manner, which might contribute to its tumor promoting effects. Since PI3K activation is linked with various cancers, combination therapy utilizing TRG and PI3K inhibitors has the potential to not only increase the efficacy of TRG as a chemotherapeutic agent but also reduce its off target effects.

Sodium hyaluronate gels as a drug‐release system for corticosteroids: release kinetics and antiproliferative potential for glaucoma surgery

To evaluate the release kinetics, biocompatibility and antiproliferative potential of a concentrated hydrophilic steroid formulation from commercially available sodium hyaluronate gels as a potential adjunct in glaucoma surgery. Dexamethasone and sodium hyaluronate 1% (Healon) and sodium hyaluronate 2.3% (Healon 5) were mixed to yield sodium hyaluronate formulations containing dexamethasone in concentrations of 4-20 mg/ml (7.7-38 mm). Non-cumulative and cumulative release into balanced salt solution (BSS) or phosphate buffered saline (PBS) was measured spectrophotometrically over 2-6 days. For cytotoxicity assays, human tenon fibroblasts (HTFB) and human retinal pigment epithelium cells (ARPE19) were cultured in a serum-deficient medium to ensure a static milieu; 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay and Live/Dead cell-mediated cytotoxicity assay were used to exclude cytotoxicity. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine (BrdU)-incorporation into cellular DNA. The release kinetics from sodium hyaluronate 1% and 2.3% were almost identical. Steady state was achieved after approximately 44 hrs in non-cumulative measurements. The release plotted as a function of the square root of time was consistent with a largely diffusion-controlled release system. No cytotoxicity could be observed. Dexamethasone-loaded sodium hyaluronate showed a significant antiproliferative effect on HTFB and ARPE19 cells. Dexamethasone-loaded sodium hyaluronate shows extended release of steroid over almost 2 days in concentrations high enough to inhibit the proliferation of HTFB and RPE cells without evoking cytotoxic effects. Thus, this formulation may be an easy-to-prepare adjunct in glaucoma surgery or other procedures in which cellular growth inhibition is desired.

Targeting Thioredoxin Reductase 1 Reduction in Cancer Cells Inhibits Self-Sufficient Growth and DNA Replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

Nucleus pulposus notochord cells secrete connective tissue growth factor and Up‐regulate proteoglycan expression by intervertebral disc chondrocytes

To identify the components of conditioned medium obtained from intervertebral disc nucleus pulposus-derived canine notochord cells, and to evaluate the capacity of such factors to affect disc-derived chondrocyte gene expression of aggrecan, versican, and hyaluronic acid synthase 2 (HAS-2) as a function of culture conditions. Canine notochord cells obtained from nonchondrodystrophic dogs were cultured within alginate beads under conditions of serum deficiency (Dulbecco's modified Eagle's medium [DMEM]) to produce notochord cell-conditioned medium (NCCM). NCCM was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectroscopy. Bovine disc-derived chondrocytes were cultured with serum-deficient medium (DMEM) and NCCM and assayed for the effect of tissue culture conditions on aggrecan, versican, and HAS-2 gene expression. Next, chondrocyte gene expression for aggrecan was evaluated using DMEM containing recombinant connective tissue growth factor (rCTGF), and the results compared with those obtained using NCCM and DMEM. NCCM contained aggrecan, Cu/Zn superoxide dismutase, fibronectin, and CTGF precursor. Culture with NCCM caused an up-regulation of aggrecan, versican, and HAS-2 gene expression. NCCM induced aggrecan gene expression in chondrocytes at a level similar to that induced by 100-200 ng/ml rCTGF. Nonchondrodystrophic and chondrodystrophic canine notochord cells exhibited similar levels of CTGF gene expression. Nucleus pulposus-derived notochord cells secrete CTGF (CCN2), a recently discovered multifunctional growth factor. There is no difference between CTGF gene expression in nonchondrodystrophic and chondrodystrophic canine notochord cells, suggesting a possible role of CTGF as an anabolic factor within the disc nucleus that is, to at least some degree, dependent on the population of notochord cells within the disc nucleus.

Secretion of Vascular Endothelial Growth Factor by Primary Human Fibroblasts at Senescence

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells

The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01–1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025%–0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically used concentrations, direct physical contact with crystalline particles might cause a local, rapid-progressive cytotoxicity that involves the induction of the apoptotic cascade. Therefore, epiretinal deposits after intravitreal TA administration might be critical in terms of long-term biocompatibility.

Nuclear sphingomyelin pathway in serum deprivation‐induced apoptosis of embryonic hippocampal cells

Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM-synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum-deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time-dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM-synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin-synthase balance in serum deprivation-induced apoptosis in the embryonic hippocampal cell is discussed.

Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G(2) phases of the cell cycle. Geminin protein levels, which are low in G(0)/G(1) and increase at the G(1)/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells

The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D 3. These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most other marker genes of osteogenic differentiation.

Conditional Expression of SV40 T-antigen in Mouse Cardiomyocytes Facilitates an Inducible Switch from Proliferation to Differentiation

Studies of cardiac muscle gene expression and signaling have been hampered by the lack of immortalized cardiomyocyte cell lines capable of proliferation and irreversible withdrawal from the cell cycle. With the goal of creating such cell lines, we generated transgenic mice using cardiac-specific cis-regulatory elements from the mouse Nkx2.5 gene to drive the expression of a simian virus 40 large T-antigen (TAg) gene flanked by sites for recombination by Cre recombinase. These transgenic mice developed tumors within the ventricular myocardium. Cells isolated from these tumors expressed cardiac markers and proliferated rapidly during serial passage in culture, without apparent senescence. However, they were unable to exit the cell cycle and failed to exhibit morphological features of terminal differentiation. Introduction of Cre recombinase to these cardiac cell lines by adenoviral delivery resulted in the elimination of TAg expression, accompanied by rapid cessation of cell division, and increase in cell size without an apparent induction of cellular differentiation. Incubation of cells lacking TAg in serum-deficient media with various pharmacological agents (norepinephrine, phenylephrine, or bone morphogenetic protein-2/4) or constitutively active calcium/calmodulin-dependent protein kinase I and/or calcineurin led to the formation of sarcomeres and up-regulation of cardiac genes involved in excitation-contraction coupling. The combination of TAg expression under the control of an early cardiac promoter and Cre-mediated recombination allowed us to derive an immortal cell line from the ventricular myocardium that could be controllably withdrawn from the cell cycle. The conditional expression of TAg in this manner permits propagation and regulated growth termination of cell types that are otherwise unable to be maintained in cell culture and may have applications for cardiac repair technologies.

Interdependence of cdk2 Activation and Interleukin-2Rα Accumulation in T Cells

We have shown previously that serum promotes T cell proliferation by acting with T cell receptor (TCR) agonists to efficiently down-regulate p27(Kip1) and activate cdk2-containing complexes. In the studies described here, the effect of serum on the expression of the alpha subunit of the interleukin-2 receptor (IL-2Ralpha) was examined. We found that serum was required for maximal and sustained IL-2Ralpha protein expression and consequent IL-2 signaling in TCR-activated splenocytes. Serum had no effect on IL-2Ralpha mRNA levels and thus modulates IL-2Ralpha expression post-transcriptionally. Unlike wild-type splenocytes, splenocytes exhibiting serum-independent cdk2 activation due to loss of p27(Kip1) efficiently expressed IL-2Ralpha in serum-deficient medium. Conversely, serum did not promote IL-2Ralpha accumulation in conditions in which cdk2 activity was blocked. These findings demonstrate that cdk2 activation is necessary and sufficient for IL-2Ralpha accumulation in TCR-stimulated splenocytes. On the other hand, IL-2 signaling was required (at least in part) for cdk2 activation in these cells. Thus, cdk2 activation, IL-2Ralpha expression, and IL-2 signaling are interdependent events, and we suggest that this feed-forward regulatory loop plays a key role in T cell mitogenesis.

P27Kip1 Regulates T Cell Proliferation

Our studies addressed the mechanism by which serum acts in conjunction with T cell receptor (TCR) agonists to promote the proliferation of primary splenic T cells. When added to resting splenocytes, TCR agonists initiated G(0)/G(1) traverse and activated cyclin D3-cdk6 complexes in a serum-independent manner. On the other hand, both TCR agonists and 10% serum were required for the activation of cyclin E-cdk2 and cyclin A-cdk2 complexes and the entry of cells into S phase. Serum facilitated cdk2 activation by maximizing the extent and extending the duration of the TCR-initiated down-regulation of the cdk2 inhibitor, p27(Kip1). Although p27(Kip1) levels were reduced (albeit submaximally) in cells stimulated in serum-deficient medium, nearly all of the cdk2 complexes in these cells contained p27(Kip1). In contrast, in cells receiving TCR agonist and 10% serum, little if any p27(Kip1) was present in cyclin-cdk2 complexes. Unlike wild-type splenocytes, p27(Kip1)-null splenocytes did not require serum for cdk2 activation or S phase entry whereas loss of the related cdk2 inhibitor, p21(Cip1), did not override the serum dependence of these responses. We also found that cdk2 activation was both necessary and sufficient for maximal expression of cdk2 protein. These studies provide a mechanistic basis for the serum dependence of T cell mitogenesis.

Anti-HLA antibody binding to HLA class I molecules induces proliferation of airway epithelial cells: A potential mechanism for bronchiolitis obliterans syndrome

Objective: Development of anti-HLA antibodies is associated with development of bronchiolitis obliterans syndrome after lung transplantation. We sought to determine the mechanism by which anti-HLA antibodies affect the development of bronchiolitis obliterans syndrome. We postulated that anti-HLA antibodies bind to the donor lung epithelium and stimulate phosphorylation and proliferation. Methods: The A549 lung epithelial carcinoma cell line was cultured in serum-deficient medium to produce static growth. Then the cells were treated with anti-HLA sera from lung transplant recipients, pooled anti-HLA serum from highly sensitized patients, or normal human serum. The cells were also treated with the W6/32 mouse anti-HLA class I monoclonal antibody or control mouse IgG. Tritiated thymidine uptake was determined at 24, 48, and 72 hours. In parallel experiments the cells were treated as described above, and the levels of tyrosine phosphorylation were determined by Western blot analysis. Results: Cells treated with anti-HLA serum or the W6/32 monoclonal antibody exhibited significantly greater proliferation and tyrosine phosphorylation of proteins of approximately 170, 130, 110, and 70 kd compared with cells treated with normal human serum or mouse IgG, respectively. Conclusions: These data indicate that anti-HLA antibodies have the ability to stimulate airway epithelial cell proliferation and that they may play an important role in the development of bronchiolitis obliterans syndrome. Prevention of HLA sensitization and immunosuppression with agents capable of blocking indirect antigen presentation and the humoral immune response against the allograft may be pivotal in preventing the development of bronchiolitis obliterans syndrome after lung transplantation. (J Thorac Cardiovasc Surg 2000;119:39-45)

Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP

Endothelial cell-derived nitric oxide (NO) has been suggested to inhibit smooth muscle cell proliferation, resulting in the reduction of intimal hyperplasia during atherogenesis. The present study investigates the role of NO from exogenous and endogenous sources on the proliferation of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (CAEC). Three different NO-generating compounds [sodium nitroprusside (SNP), S-nitroso-glutathione (GSNO) and S-nitroso-acetylpenicillamine (SNAP)] were found to inhibit endothelial cell proliferation measured with three independent methods (cell counting, [ 3H]thymidine incorporation, DNA histograms) with significant inhibition occurring at concentrations ≥100 μM. Growth-inhibiting effects were observed after long-term treatment (18–96 h) as well as after short stimulation with NO donors (10 min with a subsequent NO donor-free culture period of 18 h) and were comparable in culture medium (20% serum, growth factor supplementation) and serum-deficient medium (1% serum). The NO donor effects were mediated by the release of NO as they were prevented by NO scavenging. Superoxide dismutase (SOD) was found not to interfere with these effects suggesting that peroxynitrite formation was unlikely to be involved. 1H-[1,2,4]Oxadiazolo[4,3,-α]quinoxalin-1-one (ODQ), a specific inhibitor of the soluble guanylate cyclase, was observed not to alter the antiproliferative effects of NO donors although it completely prevented NO-mediated increase of cyclic guanosine 3′,5′-monophosphate (cGMP), suggesting that the NO-induced growth inhibition was not mediated by cGMP. Furthermore, inhibition of endogenous NO production by N-nitro- l-arginine methylester ( l-NAME) did not affect endothelial cell growth regardless of using serum plus growth factor supplement, growth factor supplement alone, or thrombin to stimulate proliferation. We suggest that constitutively synthesized NO may not regulate endothelial cell proliferation whereas the growth-inhibiting NO effects may occur when an inducible NO synthase associated with a persistently high NO production is expressed in the atherosclerotic vessel wall.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

Induced PAI-1 mRNA expression and targeted protein accumulation are early G1 events in serum-stimulated rat kidney cells.

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the SERPIN gene family that functions to regulate the plasmin-based pericellular proteolytic cascade, is growth state-regulated in normal rat kidney (NRK) cells (Ryan and Higgins, 1990, J. Cell. Physiol., 155:376-384; Ryan et al., 1996, Biochem. J., 314:1041-1046). Comparative analysis of arrest states induced in NRK cells upon exposure to serum-deficient (0.5% FBS) or serum-free culture conditions served to define the kinetics of PAI-1 gene expression and fate of de novo-synthesized PAI-1 protein. While cells rendered quiescent in serum-free or serum-deficient media were equivalent with regard to the time course of PAI-1 mRNA induction, the level of expressed transcripts (27-fold vs. 12-fold) and accumulated saponin fraction PAI-1 protein (12-fold vs. 6-fold) were consistently greater in cells recruited into exponential growth phase from a serum-free as compared to a serum-deficient arrest condition. Relative PAI-1 mRNA abundance increased within 1-2 hr post-serum addition, was maximal at 4 hr, and declined rapidly thereafter; this time course of expression coupled with placement of entry into DNA synthetic phase at approximately 12 hr after stimulation indicates that PAI-1 induction is an early-to-mid G1 phase event. Induced PAI-1 protein was evident immunocytochemically within 2 hr of serum stimulation as a peripheral "rim" of accumulated protein restricted to the cellular ventral surface at the plane of the substrate. No PAI-1 was detected between individual cells suggesting that this protein may be targeted directly to the undersurface region. By 6 hr post-stimulation, the rim of PAI-1 deposition increased in intensity and broadened to occupy approximately 30 to 50% of the total undersurface area. Double-label immunocytochemistry indicated that accumulated PAI-1 was deposited in close proximity to, but not actually within, vinculin-containing focal contact structures. Potential functionality of induced PAI-1 expression to either the initiation or maintenance of the serum-stimulated phenotype was assessed using antibodies to PAI-1. The IgG fractions of two different antisera which neutralize the ability of PAI-1 to complex with and thereby inhibit the catalytic activity of urokinase plasminogen activator significantly reduced (by 25-35%) the incidence of cells displaying the serum-stimulated phenotype; antibodies that bind PAI-1 but do not block PAI-1 inhibitory activity were without effect. In view of the vagaries of antibody accessibility and in situ neutralizing activity (particularly in a region as structurally complex as the focal contact), these data may actually underestimate the importance of PAI-1 in maintaining the activated phenotype.

Influence of cell culture medium on the photosensitizing effectiveness of bacteriochlorin a

To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich environment, the photosensitizing efficacy was tested by clonogenic survival of Chinese hamster ovary and T24 (human bladder carcinoma) cells. Confluent cell layers were incubated with 2.5 μg ml −1 BCA in cell culture medium for 1, 4, 6, 18 and 24 h. Upon illumination with red light it was found that BCA was not effective as a photosensitizer in this medium. Extraction methods showed that this lack of photosensitization could not be explained by the inability of the dye to enter the cells in the presence of cell culture medium. The presence of cell culture medium did not change the spectral properties of BCA to an appreciable extent. Standard KBr density gradient ultracentrifugation showed that in the presence of cell culture medium approximately 20% of the BCA was sedimented with low density lipoprotein (LDL) and 60% with high density lipoprotein (HDL). Incubating T24 cells 18 h before the clonogenic cell survival assay in serum-deficient medium restored the photosensitizing properties of BCA. It is proposed that in a protein-rich (in vivo) environment BCA associates with lipoproteins and can be taken up by malignant neoplasms via the LDL pathway.

Effect of bradykinin on intracellular signalling systems in a rat clonal dental pulp-cell line

The cloned pulp-cell line RDP4-1 increases cAMP production, hydrolyses phosphoinositide (PI) and mobilizes calcium in response to prostaglandin E 2 (PGE 2) and PGF 2-α. The effect of bradykinin (BK) on intracellular signalling systems and DNA synthesis was studied in these cells. BK (10 μM) transiently increased cytoplasmic free calcium ([ Ca 2+] i) both in the presence and absence of external calcium. After stimulation with BK (10 μM), cells did not respond significantly to PGE 2 (0.5 μg/ml). Pretreatment with indomethacin (30 μM) inhibited the [ Ca 2+] i increment by BK (10 μM), but not by the subsequent addition of PGE 2 (0.5 μg/ml). Also, pretreatment with PGE 2 (0.5 μg/ml) blocked the action of BK (10 μM). BK (0.1–100 μM) stimulated PI hydrolysis and cAMP production in a dose-dependent manner. Both the PI and the cAMP responses were inhibited by indomethacin (30 μM), as was the calcium response. BK (0.01–10 μM) also stimulated release of arachidonic acid and its metabolites dose-dependently. However, prolonged exposure to BK in serum-deficient medium did not exert any effect on DNA synthesis. RDP 4-1 cells, therefore, appear to respond to BK with increased cAMP production, PI hydrolysis and calcium mobilization. The inhibition of these effects of BK by indomethacin raises the possibility that cyclo-oxygenase product(s), especially PGE 2 or PGE 2-like compounds, may be responsible for evoking these effects. These results indicate that BK may stimulate or modulate cell metabolism in the dental pulp.

Apoptosis of murine BW 5147 thymoma cells induced by cold shock.

Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.