Growth Of Lung Adenocarcinoma Cells Research Articles

Identification and Preliminary Characterization of Novel Small Molecules That Inhibit Growth of Human Lung Adenocarcinoma Cells

Drug treatment for human lung cancers remains unsatisfactory, despite the identification of many potential therapeutic targets (such as mutant KRAS protein) and the approval of agents that inhibit the tyrosine kinase activity of mutant epidermal growth factor receptor (EGFR). To seek new therapeutic strategies against lung tumors, the authors have screened 189,290 small molecules for their ability to retard growth of human lung adenocarcinoma cell lines, which harbor mutations in EGFR or KRAS. Four candidates that are structurally different from common tyrosine kinase inhibitors were selected for further study. The authors describe one small molecule (designated lung cancer screen-1 [LCS-1]) in detail here. Identification of the targets of LCS-1 and other growth inhibitors found in this screen may help to develop new agents for the treatment of lung adenocarcinomas, including those driven by mutant EGFR and KRAS.

Vascular endothelial growth factor receptor targeted RNA interference inhibits growth of human lung adenocarcinoma cells

Objective:To investigate the inhibitory effects of RNA silencing via adenovirus-mediated vascular endothelial growth factor receptor (VEGFR) shRNA on proliferation of lung adenocarcinoma cells in vitro and in vivo.Methods: Ad-VEGFRshRNA adenovirus containing enhanced green fluorescent protein (EGFP) gene and VEGFRshRNA was constructed and was used to infect A549 cells; fluorescent microscopy was used to observe the infection efficiency. Western blotting assay was used to examine the expression of VEGFR protein in A549 cells. MTT method was used to examine the cell viability and the cell growth curve was drawn. The inhibition of cell growth was examined by cell cycle and colony-forming test. Meanwhile, nude mice were transplanted with A549 cells to establish tumor-bearing model, and the long term growth of tumor was observed.Results: Western blotting revealed that the expression of VEGFR was obviously decreased in the RNA interference group. The cell growth curve indicated that the cell growth was obviously inhibited after RNA interference. Cell cycle and colony-forming test indicated that the tumor growth was obviously inhibited after RNA interference. In vivo study with nude mice also indicated that RNA interference obviously inhibited tumor growth.Conclusion: The constructed VEGFR-targeted shRNA can effectively inhibit VEGFR expression in A549 cells and can suppress the growth of A549 cells.

Restoration of SOCS3 suppresses human lung adenocarcinoma cell growth by downregulating activation of Erk1/2, Akt apart from STAT3

SOCS3 is regarded as a major negative regulator of STAT3. Recent evidence indicates that SOCS3 regulates strength and duration of other signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in non-malignant cells. The repression or silence of SOCS3 expression in a few tumor types has led to speculation that loss of SOCS3 gene is closely related to deregulation of multiple signal pathways during tumorigenesis. However, apart from STAT3, little is known in malignant cells about the mechanism by which SOCS3 modulates other intracellular signal cascades such as Erk1/2 and Akt, whose aberrant activation has been implicated in many human tumors. Expression of SOCS3 proved deficient in human lung adenocarcinoma A549 cells, and forced expression of SOCS3 resulted in growth inhibition. Growth suppression due to SOCS3 was associated with attenuated activation of Erk1/2, Akt as well as STAT3. The results suggested that SOCS3, as negative regulators of cytokine signaling, might maintain homeostasis by regulating multiple signaling pathways and reverse cell malignant behavior.

Aryl hydrocarbon receptor activation and overexpression upregulated fibroblast growth factor‐9 in human lung adenocarcinomas

We had previously reported that aryl hydrocarbon receptors (AhRs) are overexpressed in lung adenocarcinomas. Benzo[a]pyrene (BaP), an AhR agonist, increased FGF-9 expression in human lung adenocarcinoma cells. Similarly, several AhR agonists increased FGF-9 mRNA levels, and BaP-induced FGF-9 expression was prevented by cotreatment with AhR antagonist in human lung adenocarcinoma cells. Furthermore, AhR agonists increased transcriptional activity of FGF-9 promoter. Modulation of AhR expression via RNA interference or transient overexpression respectively reduced or increased both constitutive and BaP-induced FGF-9 expression in human lung cells. These results suggested that AhR activation and overexpression increased FGF-9 expression in lung cells. FGF-9 increased growth of lung fibroblasts but not that of lung adenocarcinoma cells. However, conditioned media collected from FGF-9-treated fibroblasts increased cell growth of lung adenocarcinoma cells. Furthermore, lung adenocarcinoma cells expressed FGF receptor 2 and cotreatment with anti-FGF receptor 2 prevented the interaction between fibroblasts and tumor cells. It is likely that FGF-9-stimulated fibroblasts secreted unknown factors, which activated FGF receptor 2 and subsequently promoted growth of lung adenocarcinoma cells. We further compared AhR and FGF-9 expression in 146 non-small cell lung cancer (NSCLC) cases by immunohistochemistry. FGF-9 expression was more common in adenocarcinomas than in squamous cell carcinomas. Furthermore, FGF-9 and AhR expression were well correlated in lung adenocarcinomas. These results suggest that AhR expression correlated positively with FGF-9 expression in lung adenocarcinomas, which might promote tumor growth by modulating communication between tumor cells and fibroblasts. Preventing AhR overexpression or disturbing FGF-9 function may reduce the development of lung adenocarcinomas. (c) 2009 UICC.

Restoration of tumour suppressor hsa-miR-145 inhibits cancer cell growth in lung adenocarcinoma patients with epidermal growth factor receptor mutation

Background In Hong Kong, about 30% of non-small cell lung cancer patients have never smoked tobacco. Among women, 83% are never-smokers and their histological type is invariably adenocarcinoma with 70% incidence of epidermal growth factor receptor ( EGFR) mutation. The present study focuses on the microRNA (miRNA) expression profiles of this important subset of lung cancer. Methods Paired samples collected from the lung cancer tissue and adjacent normal lung parenchyma of 10 non-smoking patients with lung adenocarcinoma were profiled by miRNA microarray. Results were validated by quantitative reverse transcription polymerase chain reaction. Transfected cell viability assays were applied to determine the effects of candidate miRNAs on lung cancer cells. Results Comparing paired lung cancer tissue with adjacent normal lung parenchyma, hsa-miR-126 ∗, hsa-miR-145, hsa-miR-21, hsa-miR-182, hsa-miR-183 and hsa-miR-210 were found to be the most differentially expressed miRNAs. Most interestingly, an obvious inhibition of cell growth was observed in the EGFR mutant lung adenocarcinoma after transfection of hsa-pre-miR-145. Conclusions Our study is the first report to connect miR-182 to lung cancer. Our results also show that restoration of tumour suppressor hsa-miR-145 inhibits cancer cell growth in EGFR mutant lung adenocarcinoma. Further study on these specific differentially expressed miRNAs may provide important information on peculiar tumourigenetic pathways and may identify useful biomarkers.

The effects of cetuximab alone and in combination with endostatin on vascular endothelial growth factor and interleukin-8 expression in human lung adenocarcinoma cells

Background: Angiogenesis, the growth of new blood vessels, plays an important role in tumor growth and metastasis. Both cetuximab and endostatin have been found to reduce the expression of endothelial-stimulating growth factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8. However, the effects of cetuximab alone or in combination with endostatin on human lung adenocarcinoma cell growth remain unclear. Objective: The aim of this study was to evaluate the cellular and molecular effects of cetuximab alone and in combination with endostatin on human lung adenocarcinoma cell lines HI 299, SPC-A1, and H460 in vitro. Methods The epidermal growth factor receptor (EGFR) status of a panel of human lung adenocarcinoma cell lines was characterized using Western blot analysis. We used a modified tetrazolium salt assay to evaluate the growth-inhibitory effects of cetuximab and endostatin alone and in combination on the cell lines. We also determined the effects of these 2 drugs on VEGF and IL-8 expression using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Cells were treated for 4 days with cetuximab 12.5 μ/mL, endostatin 25 μ/mL, or cetuximab 12.5 μg/mL + endostatin 25 μg/mL. Untreated cells cultured for 4 days served as controls. Results: EGFR expression in the H1299 cells was higher than in the SPC-A1 and H460 cells. Varying concentrations of cetuximab alone were associated with a significant growth-inhibitory effect on all 3 cell lines in a dose-dependent manner after 4 days of exposure compared with controls (all, P < 0.05). Compared with controls, varying concentrations of endostatin alone were not associated with significant inhibition of cell growth in any of the 3 cell lines. The inhibitory ratio of cetuximab + endostatin at varying concentrations was significantly greater than that of cetuximab alone (all, P < 0.05). On ELISA, either drug alone was associated with significant reductions in secreted VEGF and IL-8 in the HI 299, SPC-A1, and H460 cell lines (all, P < 0.05), with the exception of IL-8 concentration in the H460 cells. Mean (SD) VEGF expression with combination treatment in the H1299 and SPC-A1 cell lines (687 [21] and 629 [23] pg/mL, respectively) was significantly lower than with cetuxi-mab alone (878 [31] and 708 [20] pg/mL; both, P < 0.001); in the H460 cell line, combination treatment was not associated with a significant further reduction in VEGF expression. IL-8 concentrations with cetuximab in the H1299, SPC-A1, and H460 cell lines were 628 (20), 484 (29), and 532 (28) pg/mL, respectively, while the IL-8 concentrations with the combination treatment were 516 (20), 480 (18), and 467 (30) pg/mL. An enhanced effect of endostatin on IL-8 was observed in the H1299 and H460 cell lines ( P < 0.001 and P = 0.018, respectively); however, no enhanced effect in the SPC-A1 line was observed. Similar results for VEGF and IL-8 expression were found using Western blot analysis. Conclusions: The results from this in vitro study suggest that cetuximab treatment might both inhibit human lung adenocarcinoma cell line growth and reduce the expression of VEGF and IL-8, which are the biomarkers of angiogenesis. Endostatin was not associated with inhibition of human lung adenocarcinoma cell line growth directly. Findings with the combination of cetuximab + endostatin suggest that endostatin might enhance the antiangiogenic and antitumor activity of cetuximab through an apparent effect on VEGF expression and, to a lesser degree, on IL-8 expression.

Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

Fibroblast growth factor (FGF)-2 is a critical growth factor in normal and malignant cell proliferation and tumor-associated angiogenesis. Fibrinogen and fibrin bind to FGF-2 and modulate FGF-2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen. Herein we examined the role of fibrinogen and FGF-2 interactions on prostate and lung adenocarcinoma cell growth in vitro. Cell proliferation was measured by (3)H-thymidine uptake and the specificity of FGF-2-fibrinogen interactions was measured using wild-type and mutant FGF-2s, fibrinogen gamma-chain (FGG) RNAi and co-immunoprecipitation. Metabolic labeling, immunopurification and fluorography demonstrated de novo fibrinogen production. FGF-2 stimulated DU-145 cell proliferation, whereas neither FGF-2 nor fibrinogen affected the growth of PC-3 or A549 cells. Fibrinogen augmented the proliferative effect of FGF-2 on DU-145 cells. The role of fibrinogen in FGF-2-enhanced DNA synthesis was confirmed using an FGF-2 mutant that exhibits no binding affinity for fibrinogen. FGG transcripts were present in PC-3, A549 and DU-145 cells, but only PC-3 and A549 cells produced detectable levels of intact protein. RNAi-mediated knockdown of FGG expression resulted in decreased production of fibrinogen protein and inhibited (3)H-thymidine uptake in A549 and PC-3 cells by 60%, which was restored by exogenously added fibrinogen. FGF-2 and fibrinogen secreted by the cells were present in the medium as a soluble complex, as determined by coimmunoprecipitation studies. These data indicate that endogenously synthesized fibrinogen promotes the growth of lung and prostate cancer cells through interaction with FGF-2.

Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.

Clarithromycin Suppresses Invasiveness of Human Lung Adenocarcinoma Cells

Background: It has been speculated that clarithromycin (CAM), a 14-membered ring macrolide, possesses antitumor effects besides antimicrobial and anti-inflammatory effects. Method: We evaluated the effects of CAM on the growth and invasiveness of A549 lung adenocarcinoma cells. Results: Although CAM did not affect the growth of A549 cells, the Matrigel invasion assay showed that the potential of invasion was diminished by CAM treatment. When analyzed by flow cytometry, CAM suppressed α₂- and β₁-integrin expression. Furthermore, thymidine phosphorylase (TP) expression was diminished by CAM treatment in a dose-dependent manner. A specific TP inhibitor also suppressed β₁-integrin expression in flow cytometric analysis. Conclusions: These results suggest that CAM may suppress invasive activity of A549 cells in part by diminishing the expression of TP, α₂- and β₁-integrin, which may be a downstream signal of the TP pathway, and that CAM could be useful in the treatment of lung adenocarcinoma.

Inhibitory effect of pulmonary carcinoma by adenovirus-mediated CD/UPRT gene

The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000 microg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

Src Tyrosine Kinase Inhibitor, M475271, Suppresses Subcutaneous Growth and Production of Lung Metastasis Via Inhibition of Proliferation, Invasion, and Vascularization of Human Lung Adenocarcinoma Cells

Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of all three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.

Growth inhibition of NS-398 combined with cisplatin on human lung adenocarcinoma cells and its mechanism

In recent years, many investigations have showed that COX-2 inhibitors not only inhibit the growth of tumor cells but also enhance the cytotoxicity of anticancer drugs, such as NS-398, a selective COX-2 inhibitor. But the effect of NS-398 combined with chemotherapy agent cisplatin on growth of lung adenocarcinoma cells and its mechanism are still unknown. The aim of this study is to investigate the effect of NS-398 combined with cisplatin on proliferation of human lung adenocarcinoma cell lines, and to explore their mechanisms. Synergistic effect of NS-398 and cisplatin on proliferation of human lung adenocarcinoma cell lines was detected by MTT growth assay, and data were analyzed by SPSS general linear models procedure to determine the IC₅₀ of each drug alone. Combined index (CI) was analyzed to determine the combined effect of drugs. Apoptosis and cell cycle distribution were detected by fluorescence staining of Acridine orange (AO) and Ethidium bromide (EB) and flow cytometry. The various concentration of NS-398 in combination with cisplatin resulted in the reduction of IC₅₀ of cisplatin by 48.25%-62.44% in A549 cells, and by 43.34%-69.61% in SPC-A-1 cells. The CI of NS-398 and cisplatin in the two cell lines ranged from 0.4 to 0.9, which indicated that they acted in slight synergistic to synergistic pattern. The apoptotic rate at 24h and 48h in the combined groups was significantly higher than those of the control groups, and the S-phase cell fractions at 48h and 72h were obviously higher than those of the control groups. There was a positive correlation between apoptotic rate and S-phase cell fraction in the combined groups (r=0.882,P= 0.01). NS-398 can improve growth inhibition of cisplatin on lung adenocarcinoma cells, and the two agents act in synergistic way. This synergistic effect may be related to apoptosis pathway.

Effect of carbonic anhydrase-related protein VIII expression on lung adenocarcinoma cell growth

Carbonic anhydrase-related protein VIII (CA-RP VIII) is expressed in most non-small cell lung cancer, and especially strongly at the front of tumor progression. Screening analysis of CA-RP VIII expression in a panel of cultured lung cancer cell lines showed that a well differentiated adenocarcinoma cell line, PC-9, appeared to lack CA-RP VIII. Subsequently, CA8 cDNA was transfected with an expression vector into PC-9. Ectopic overexpression of CA-RP VIII reduced the growth of PC-9 cells on uncoated culture dishes, especially when the cultures were started at low cell density, but increased cell growth on laminin-coated dishes. Interestingly, ectopic CA-RP VIII expression markedly reduced caspase-3 activity induced by serum starvation and anti-cancer agents in PC-9 cells. The present findings suggest that CA-RP VIII expression promotes progression of lung cancer by multifarious mechanisms.

Role of thymidine phosphorylase on invasiveness and metastasis in lung adenocarcinoma.

Thymidine phosphorylase (TP) is expressed at higher levels in a variety of human cancers than in adjacent normal tissue. It is reported that the higher expression is associated with an increase of intratumoral microvessel density and a poor prognosis. We investigated the role of TP in human non small cell lung cancers (NSCLCs). The concentrations of TP in the tumors and the adjacent normal tissue from surgically resected specimens of 54 cases of NSCLCs were measured by using an enzyme-linked immunosorbent assay. Tumor specimens were also examined immunohistochemically. TP concentrations in the tumors were 169 +/- 18 units/mg protein (mean +/- SD), whereas those in normal tissue were 43 +/- 4 units/mg protein (mean +/- SD), consistent with TP staining patterns. There was no correlation between TP expression and microvessel density. Among clinicopathologic factors examined, the concentrations of TP but not TP immunoreactivity correlated with tumor differentiation in lung adenocarcinoma. Although a specific TP inhibitor (TPI) and overexpression of TP did not affect the growth of A549 lung adenocarcinoma cells, Matrigel invasion assay showed that A549 transfected with TP had higher invasive potential than mock transfectant, and such enhanced invasive activity was markedly diminished by treatment with TPI. Furthermore, administration of TPI suppressed lung metastasis of TP-overexpressing A549 cells in nude mice. These results demonstrate that TP may play an important role in tumor differentiation, invasiveness and metastasis in lung adenocarcinoma, and suggest that TP could be a novel target for treatment of TP-overexpressing lung adenocarcinoma.

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells.

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28). It is noteworthy that high inhibitory effects of the three-component hybrid liposomes composed of DMPC, Tween 20, and LacCn (DMPC:Tween 20:LacCn=65:7:28) on the growth of glioma (U251) and lung adenocarcinoma (RERF-LC-OK) cells were attained in vitro without any drug, although no significant inhibitory effects of any individual component (DMPC, Tween 20, LacCn) or the two-component hybrid liposomes of DMPC and Tween 20 on the growth of tumor cells examined were obtained.

Activation of Hepatocyte Growth Factor-Met Autocrine Loop Enhances Tumorigenicity in a Human Lung Adenocarcinoma Cell Line

Hepatocyte growth factor (HGF) is a multifunctional cytokine with effects on the proliferation, motility, and differentiation of cells that express its receptor Met. The co-expression of HGF and Met is common among nonsmall-cell lung cancers, especially adenocarcinoma. However, the biologic consequences of this putative HGF-Met autocrine signaling remain speculative. We have used retroviral gene transduction technique to express high levels of HGF in the NCI-H358 lung adenocarcinoma cells that have functionally active cell surface Met receptor. The activation of autocrine HGFMet signaling was confirmed by the induction of spontaneous cell scattering activity. Compared to the parent and control cells transduced with the retroviral vector alone, HGF overexpressing H358 cells show enhanced capacity to colonize soft agar medium and to form xenograft tumors when implanted in the subcutaneous tissue of immune-deficient mice. These effects were not accompanied by changes in their growth rate in monolayer culture condition, or in the expression of vascular endothelial growth factor. The tumors formed by HGF overexpressing cells also showed more prominent glandular cell arrangement and functional activity. This report provides the direct in vivo evidence that autocrine HGF-Met signaling plays significant roles in the growth and differentiation of human lung adenocarcinoma cells.

Remarkably enhanced inhibitory effects of hybrid liposomes on the growth of specific tumor cells

The highly specific inhibitory effects of the hybrid liposomes composed of 90 mol% l-α-dimyristoylphosphatidylglycerol and 10 mol% polyoxyethylene (10) dodecyl ether on the growth of lung adenocarcinoma and stomach tumor cells in vitro were obtained for the first time.

The role of recombinant Rb gene adenovirus vector in the growth of lung adenocarcinoma cells

Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy.

Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction. Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined. For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required. The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space. Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac. Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease. In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells. The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol. We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.

Modulation of extracellular proteolytic activity and anchorage-independent growth of cultured cells by sarcoma cell-derived factors: Relationships to transforming growth factor-β

We have previously described a factor(s) produced by 8387 fibrosarcoma cells, which can affect plasminogen activator (PA) activity of cultured cells. Since then, transforming growth factor-β (TGFβ) has, been established as a major growth factor/growth inhibitor that regulates both the expression and activity of PAs and their endothelial-type inhibitor (PAI-1). The present study was undertaken to characterize the 8387 fibrosarcoma cell-derived factor(s) and to investigate its relationships to TGFβ by analysis of modulation of PA activity and cell growth. The fibrosarcoma cell-derived proteins were partially purified from serum-free conditioned culture medium using Bio-Gel P-10 chromatography. Two separate fractions with apparent molecular weights of 16,000 and 12,000 contained activities that both decreased the secretion of PA activity by human lung fibroblasts and inhibited the soft agar growth of A549 lung adenocarcinoma cells. Both factors affected similarly the production of urokinase-type PA and PAI-1 in various cell lines and enhanced anchorage-independent growth of murine AKR-2B fibroblasts. The effects of these factors thus resembled those of TGFβ. The immunological relationships between the M r 16,000 and M r 12,000 factors and TGFβ were therefore studied using neutralizing anti-TGFβ antibodies. The TGFβ antibodies efficiently inhibited the effects of the M r 16,000 factor but not those of the M r 12,000 factor in cell culture assays. The results suggest that 8387 fibrosarcoma cells produce two major growth inhibitors, one of which is closely related to TGFβ.