Bronchioloalveolar Carcinoma Cell Research Articles

Imaging of bronchioloalveolar carcinoma in the mice with the αⅤβ3 integrin-targeted tracer 99mTc-RGD-4CK

Receptor-based imaging agents have shown improved specificity and sensitivity of cancer diagnosis by targeting the specific features of cancer. Here we reported the (99m)Tc-labeling of a cyclic polypeptide RGD-4CK and the characterization of this agent in vitro and in bronchioloalveolar carcinoma (BAC) xenograft model. The αⅤβ3 integrin receptor binding affinity of (99m)Tc-RGD-4CK was determined in BAC. The cancer targeting properties of (99m)Tc-RGD-4CK were determined in NCI-H358 xenografted nude mice. Moreover, the BAC uptake of (99m)Tc-RGD-4CK was blocked with nonradiolabeled RGD-4CK in xenografts. The competitive assay showed that (99m)Tc-RGD-4CK exhibited high specificity to BAC cell line NCI-H358. Biodistribution studies indicated that (99m)Tc-RGD-4CK exhibited high tumor uptake (4.12 ± 1.21% injected dose/g 120 minutes after injection) and prolonged tumor retention (2.08 ± 0.33% injected dose/g 240 minutes after injection) in NCI-H358 xenografted nude mice. Moreover, (99m)Tc-RGD-4CK produced a good tumor-to-lung ratio (2.38) because of low lung activity accumulation 120 minutes postinjection. BAC on the flank of xenografted mice was clearly visualized by single photon emission computed tomography/computed tomography imaging using (99m)Tc-RGD-4CK. In conclusion, this study provides evidence that (99m)Tc-RGD-4CK is a promising agent for noninvasive determination of αⅤβ3 integrin status and therapy monitoring in BAC.

The role of Necl-5 in the invasive activity of lung adenocarcinoma

Nectin-like molucule-5 (Necl-5) is an immunoglobulin-like molecule that was originally identified as a poliovirus receptor and is often upregulated in cancer cells. It has been said that Necl-5 plays a role in not only cell–cell adhesion, but also cell migration, proliferation, and metastasis. In this study, we used a bronchioloalveolar carcinoma (BAC) cell line and fibroblasts to assess the expression of Necl-5 in the development of cancer–stroma communication by using an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that enables visualization of cell migration during invasion. The expression of Necl-5 was higher in BAC cells than in fibroblasts. This tendency didn't change even when the BAC cells were mixed with fibroblasts. To assess the role of Necl-5 in the invasive activity of the BAC cells, we knocked down its expression using RNA interference (RNAi). The invasion assay with DL-CGH revealed that inhibition of Necl-5 expression in the BAC cells was associated with suppressed invasiveness. In addition, Necl-5 knockdown inhibited the movement and proliferation of the BAC cells. Necl-5 expression in lung cancer cells is crucial for their invasiveness in the cancer–stromal interaction, suggesting that Necl-5 could be a favorable molecular target for the suppression of invasiveness in lung adenocarcinoma.

Abstract 2232: Overexpression of the mu opioid receptor in human non-small cell lung cancer promotes Akt and mTOR activation, tumor growth and metastasis

Abstract Background: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that mu opioid agonists can influence cancer progression. Based on our previous studies indicating the mu opioid receptor (MOR) is upregulated in several types of non-small cell lung cancer (NSCLC), this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. Methods: Stable vector control (VC) and MOR1 overexpressing human NSCLC bronchioloalveolar carcinoma (BAC) cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus) or the peripheral MOR antagonist, methylnaltrexone (MNTX). In human NSCLC xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. Results: We provide evidence that MOR is an important regulator of lung cancer progression, a disease with high mortality and few treatment options. MOR overexpression increased Akt and mTOR activation, proliferation and extravasation in human BAC cells. In vivo, overexpression of MOR in BAC increased primary tumor growth rates in nude mice by ∼2.5 fold and lung metastasis by ∼20 fold compared to VC BAC cells. Conclusions: Our overexpression data suggests a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. Our observations further suggest that exploration of MOR in NSCLC merits further study both as a diagnostic and therapeutic option. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2232. doi:1538-7445.AM2012-2232

Overexpression of the μ-Opioid Receptor in Human Non-Small Cell Lung Cancer Promotes Akt and mTOR Activation, Tumor Growth, and Metastasis

Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that μ-opioid agonists can influence cancer progression. Based on our previous studies indicating the μ-opioid receptor (MOR) is up-regulated in several types of non-small cell lung cancer, this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. Stable vector control and MOR1 overexpressing human bronchioloalveolar carcinoma cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus), or the peripheral MOR antagonist, methylnaltrexone. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in lungs of the nude mouse model. The authors provide evidence that MOR is an important regulator of lung cancer progression. MOR overexpression increased Akt and mTOR activation, proliferation, and extravasation in human bronchioloalveolar carcinoma cells. In vivo, overexpression of MOR in human bronchoalveolar carcinoma cells increased primary tumor growth rates in nude mice by approximately 2.5-fold and lung metastasis by approximately 20-fold compared with vector control cells (n = 4 per condition). The overexpression data suggest a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. The authors' observations further suggest that exploration of MOR in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Small non-mucinous bronchioloalveolar carcinoma (BAC) with ALK immunoreactivity. Immunohistochemistry for ALK showing strong expression in the cytoplasm of small non-mucinous BAC cells.

Small non-mucinous bronchioloalveolar carcinoma (BAC) with ALK immunoreactivity. Immunohistochemistry for ALK showing strong expression in the cytoplasm of small non-mucinous BAC cells.

Relationship of aquaporin 1, 3, and 5 expression in lung cancer cells to cellular differentiation, invasive growth, and metastasis potential

An oncogenic capacity of aquaporins, transmembrane channels for water, was recently proposed. This study seeks to elucidate the involvement of aquaporin 1, 3, and 5 in the development and progression of lung cancer. Expression of aquaporin 1, 3, and 5 was examined by immunohistochemistry, Western blot, and laser-captured microdissection/real-time reverse transcription polymerase chain reaction in 160 lung cancers of various histologic subtypes; and its correlation with clinicopathological factors and survival was analyzed. Aquaporin 1, 3, and 5 were expressed in tumor cells in 71%, 40%, and 56% of lung cancers, respectively. Aquaporin expressions were frequent in adenocarcinomas, whereas aquaporin 1 and 5 were negative in squamous cell carcinomas. Bronchioloalveolar carcinoma cells exhibited an apicolateral aquaporin 1 and apicolateral or basolateral aquaporin 3 localization in nonmucinous type, and apical aquaporin 1 and 5 and basolateral aquaporin 3 expression in mucinous type, which corresponded to aquaporins expression of nonneoplastic lung tissue. Basolateral aquaporin 5 expression was acquired during tumorigenesis of nonmucinous bronchioloalveolar carcinoma. In contrast, invasive adenocarcinoma tumor cells overexpressed aquaporin 1 and 5 with loss of subcellular polarization and with an intracytoplasmic distribution. Overexpression of aquaporin 1 correlated with high postoperative adenocarcinoma metastasis ratios and unfavorable disease-free survival rates (P = .031). We conclude that expression patterns of aquaporin 1, 3, and 5 in lung cancer cells are mostly associated with cellular differentiation. However, the expression of aquaporin 1 and 5 is up-regulated in invading lung cancer cells, particularly in adenocarcinomas; and the overexpression of aquaporin 1 with loss of subcellular polarization is suggested to be involved in their invasive and metastatic potential.

Abstract 5154: Human umbilical cord matrix stem cells transfected with the interferon-β gene markedly attenuated the growth of bronchioloalveolar carcinoma xenografts in mice

Abstract Mesenchymal stem cells derived from the human umbilical cord matrix (UCMSC) have great potential for therapeutic use in multiple diseases including cancer. UCMSC can be used as carriers for targeted delivery of genes or therapeutics and for local production of biologic agents. The therapeutic strategy using UCMSC as cellular vehicles for targeted gene delivery can avoid the problem of short life or excessive toxicity of a particular gene product in vivo. Interferon-β (IFN-β) has demonstrated a potent anti-tumor effect on many tumor cell lines in vitro. However, in vivo inhibition of tumor cell growth by IFN-β required blood concentrations much higher than the maximally tolerated dose. Therefore, clinical therapy by systemic administration of IFN-β was not possible. The aim of this study was to determine the anti-cancer effect of IFN-β gene transfected-UCMSC (IFN-UCMSC) on bronchioloalveolar carcinoma (BAC) cell lines in vitro and in vivo. The conditioned medium derived from IFN-UCMSC significantly attenuated cell growth of H358 and SW1573 BAC cell lines in vitro compared to the medium derived from non-transfected UCMSC. The percentage of dead cells, as measured by the trypan blue exclusion test, was significantly higher in the cells treated with the medium derived from IFN-UCMSC than UCMSC alone. The co-culture of IFN-UCMSC with cancer cells inhibited cell growth significantly through stimulation of apoptosis. Finally, systemic administration of IFN-UCMSC (0.3 million cells/injection, 5 day interval for four times) markedly attenuated tumor multiplicity and size of orthotopic H358 BAC xenografts in SCID mouse lungs. Histochemical analysis of the tumors indicated tumor-targeted localization of UCMSC and significant reduction of tumor cell proliferation index as measured by Ki-67 staining. These results clearly indicated that IFN-β production by IFN-UCMSC attenuated growth of BAC cells by inducing programmed cell death both in vitro and in vivo. This research clearly demonstrated that IFN-UCMSC are a powerful anti-cancer therapeutic tool as delivery vehicles for IFN-β. This work was supported by the KSU Terry C. Johnson Center for Basic Cancer Research, KSU Provost's Fund, KSU CVM Dean's Fund, NIH P20 RR017686 and a Joan's Legacy Research Grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5154.

Human umbilical cord matrix-derived stem cells expressing interferon-β gene significantly attenuate bronchioloalveolar carcinoma xenografts in SCID mice

Mesenchymal stem cells derived from the human umbilical cord matrix (hUCMSCs) have great potential for therapeutic use for multiple diseases. The strategy that uses therapeutic gene-transfected hUCMSCs as cellular vehicles for targeted biologic agent delivery has solved the problem of short half-life or excessive toxicity of biological agent(s) in vivo. Interferon-β (IFN-β) has demonstrated a potent antitumor effect on many types of cancer cell lines in vivo. The aim of this study was to determine the anti-cancer effect of IFN-β gene-transfected hUCMSCs (IFN-β-hUCMSCs) on cells derived from bronchioloalveolar carcinoma, a subset of lung adenocarcinoma that is difficult to treat. The co-culture of a small number of IFN-β-hUCMSCs with the human bronchioloalveolar carcinoma cell lines H358 or SW1573 significantly inhibited growth of both types of carcinoma cell lines. The culture medium conditioned by these cells also significantly attenuated the growth of both carcinoma cells, but this attenuation was abolished by adding anti-IFN-β antibody. Finally, systemic administration of IFN-β-hUCMSCs through the tail vein markedly attenuated growth of orthotopic H358 bronchioloalveolar carcinoma xenografts in SCID mice by increasing apoptosis. These results clearly indicate that IFN-β-hUCMSCs caused cell death of bronchioloalveolar carcinoma cells through IFN-β production, thereby attenuating tumor growth in vivo. These results indicate that IFN-β-hUCMSCs are a powerful anti-cancer cytotherapeutic tool for bronchioloalveolar carcinoma.

The Role of Pemetrexed in Lung Adenocarcinoma, Mixed Subtype with Bronchioloalveolar Carcinoma Features

Bronchioloalveolar carcinoma (BAC) and adenocarcinoma with bronchioloalveolar features (ADC-WBF) belong to the same anatomo-clinical entity and show very similar epidemiologic, clinical and biological characteristics. However there is a lack of consensus for the treatment of unresectable forms. Although epidermal growth factor receptor tyrosine kinase inhibitors and paclitaxel do have some efficacy, the aim of this review is to assess the role that pemetrexed, a new generation antifolate, could play in this context. Pemetrexed has proved to be particularly effective in advanced lung adenocarcinomas and unresectable mesotheliomas, and cases of major and sustained responses of ADC-WBF to pemetrexed have been reported. The preclinical rationale explaining this efficacy is that it inhibits the growth of BAC cell lines in vitro. BAC tumors overexpress FR-alpha, a protein involved in pemetrexed intracellular transport, at rates higher than those observed in lung adenocarcinomas and mesotheliomas and it would seem that pemetrexed efficacy is correlated to FR-alpha expression. The role played by thymidylate synthase expression level in sensitivity to pemetrexed also needs to be specifically explored in ADC-WBF. The results of two phase II trials, SWOG 0526 and IFCT 05-04, will hopefully provide decisive information on the relevance of pemetrexed in ADC-WBF management and the molecular predictors of response.

Abstract C79: Methylnaltrexone inhibits EGF- and IGF-induced human bronchioloalveolar carcinoma proliferation and migration

Abstract Introduction: Non-small cell lung cancer (NSCLC) has a very poor prognosis and improved therapies are needed. Expression of the mu opioid receptor (MOR) is increased in metastatic sites of patients with NSCLC. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Wang et al., Anticancer Res, 2009; Mol Cancer Ther, 2008; Gupta et al., Cancer Res, 2002). In this study, we investigated whether inhibition of MOR by the peripheral MOR antagonist, methylnaltrexone (MNTX), attenuates EGF- and IGF-mediated human bronchioloalveolar carcinoma (BAC) molecular pathways involved in NSCLC oncogenic potential. Methods: Human H358 BAC cells were pretreated with various siRNAs targeting MOR, Akt, cortactin, Gab1, PLC 1 or STAT3 or pretreated with MNTX (1.0–250 nM), 10-(4 -(N-diethylamino)butyl)-2-chlorophenoxazine (Akt inhibitor, 5 µM), U-73122 (PLC inhibitor, 1 µM) or 6-Nitrobenzo[b]thiophene-1,1-dioxide (STAT3 inhibitor, 1 µM) prior to the addition of EGF or IGF (10 ng/ml). Functional (cell proliferation and migration) and biochemical studies (immunoprecipitation, immunoblotting) were then conducted. Results: 14 NSCLC cell lines (7 adenocarcinomas, 3 SCC, 2 bronchoioloalveolar carcinoma, 1 large cell carcinoma, 1 adenosquamous carcinoma) were examined for MOR expression using immunoblotting techniques. MOR expression was increased in most NSCLC cell lines with BAC having the highest levels of MOR expression (7–9 fold higher that control primary lung epithelial or BEAS-2B cells). EGF and IGF treatment of BAC (10 ng/ml, 5–30 minutes) stimulated MOR association with the EGF and IGF receptor, respectively. MNTX inhibited EGF- and IGF-mediated BAC, but not control BEAS-2B, proliferation and migration in a dose-dependent manner (IC50 = ∼10 and 100 nM). On a mechanistic level, we observed that MNTX inhibited both EGF- and IGF-induced recruitment of the adaper protein, Gab1 (GRB2-associated binding protein 1), to the plasma membrane and complex formation with the EGF/IGF receptor in BAC. Silencing (siRNA) MOR, Gab1 or MNTX treatment inhibited EGF/IGF-mediated Akt (serine/threonine kinase), cortactin (actin regulatory protein), PLC 1 (phospholipase) and STAT3 (Signal Transducer and Activator of Transcription protein) phosphorylation/activation, events which are involved in BAC oncogenic signaling. In addition, inhibiting (siRNA and/or chemical inhibitors) Akt, cortactin, PLC 1 and/or STAT3 attenuated both EGF- and IGF-induced BAC growth and migration (50–90%). Conclusion: We have shown that MNTX inhibits EGF/IGF-mediated oncogenic signaling through inhibition of MOR-mediated Gab1 recruitment to the plasma membrane and consequent attenuation of Akt, cortactin, PLC 1 and STAT3 activation leading to reduced BAC proliferation and migration. Further study of this molecule as a potential therapeutic agent is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C79.

Short-Term Gefitinib Treatment Brought About a Long-Term Regression of Bronchioloalveolar Carcinoma Without EGFR Gene Alterations: A Case Report

The tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) gefitinib has beneficial effect in some patients with refractory advanced non-small cell lung cancer (NSCLC). However, the majority of responders eventually develop acquired resistance during the course of prolonged continuous treatment. Here we present a case of 76-year-old Japanese female, who had never smoked, with poor performance status from bronchioloalveolar carcinoma (BAC), in whom a brief initial 5-week administration of gefitinib resulted in dramatic antitumor effects that lasted approximately 8.5 months after cessation of the treatment. Furthermore, the relapsed tumor later regressed again by re-treatment with the TKI. She survived 26 months since she first took gefitinib. Unexpectedly, neither sensitizing mutations for EGFR-TKIs nor increased copy numbers were detected in EGFR gene of her BAC cells. This case suggests that, in some patients with NSCLC, even short-term administration of gefitinib may bring about clinical benefits and disease response comparable to the standard long-term daily dosing schedule. Short-term use of gefitinib will also be able to minimize the expensive medical cost of the TKI. The potential role of short-term or pulse-dose therapy with EGFR-TKIs should be clarified in further prospective studies. Moreover, it is urgent to develop better strategies by which we could distinguish responders to the TKIs from nonresponders among patients who do not have any EGFR gene alterations.

Induction of epithelial‐mesenchymal transition‐related genes by benzo[a]pyrene in lung cancer cells

It is believed that epithelial-mesenchymal transition (EMT) occurs during the development and progression of cancer; however, the correlation between tobacco smoking and EMT remains to be elucidated. Cells from the bronchioloalveolar carcinoma cell line A549 were exposed to benzo(a)pyrene (B[a]P) for 24 weeks, and morphology, proliferative activity, and gene expression profiles were analyzed. Although no apparent morphologic changes were observed, the B[a]P-exposed A549 cells exhibited enhanced proliferative activity in 1% bovine serum that contained medium, and dramatic changes in expression levels were observed in a large number of genes. Of those, the expression of EMT-related genes, such as migration-stimulating factor, plasminogen activator inhibitor-1, fibronectin, twist, transforming growth factor-beta2, basic fibroblast growth factor, and electron transport system, were up-regulated; whereas gene expression of E-cadherin was decreased. Most enhanced expression levels remained 8 weeks after the retrieval of B[a]P in culture. The current results indicated that B[a]P seems to induce EMT in lung cancer cells, and it also may drive disease progression in patients with lung cancer.

Electrophoretic mobility shift assay on poly(ethylene glycol)‐modified glass microchips for the study of estrogen responsive element binding

The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.

Novel peptide ligands for integrin α4β1 overexpressed in cancer cells

Abstract Using the “one-bead one-peptide” combinatorial technology, a library of random cyclic octapeptides and nonapeptides, consisting of natural and unnatural amino acids, was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment and proliferation of bronchioloalveolar carcinoma cells (H1650), employing a “cell growth on bead” assay. Consensus peptide sequences of cNleDXXXXc and cXNleDXXXXc (where Nle is norleucine) were identified. With alanine scanning and site-directed deletion, a typical ligand consisted of a motif of -NleDI/V/Nle- with two flanking cysteines. These peptide ligands were specific for promoting cell attachment of the H1650 cells and the cells of lymphoid cancers (Jurkat and Raji) but not other selected human cell lines of lung cancer and fibroblast. In an antibody blocking assay, integrin α4β1, which was overexpressed in H1650, Jurkat, and Raji, was identified as a putative receptor for these peptide ligands. Using Chinese hamster ovary cells transfected with either wild-type or mutant integrin α4, a critical binding site for these peptides was localized to the glycine residue at position 190 of integrin α4.

Stimulation of vascular endothelial growth factor gene transcription by all trans retinoic acid through Sp1 and Sp3 sites in human bronchioloalveolar carcinoma cells.

In this study, we examined the effects of all trans-retinoic acid (at-RA) on the vascular endothelial growth factor (VEGF) expression in human bronchioloalveolar carcinoma NCI-H322 cells to evaluate the potential of at-RA to affect tumor progression. Northern blot and enzyme-linked immunosorbent assay analyses indicate that VEGF production is significantly increased by 1 microM of at-RA. A series of 5'-deletion and site-directed mutation analyses indicated that G+C-rich sequence located at -81 and -52 was required for at-RA- and retinoic acid receptor alpha-mediated induction of VEGF promoter. Electrophoretic mobility shift and supershift assays showed that major constituents of nuclear factors binding to G+C-rich sequences are Sp1 and Sp3. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the at-RA-mediated induction of VEGF mRNA expression. Likewise, at-RA-mediated VEGF expression was completely blocked in the presence of genistein, an inhibitor for tyrosine kinases. These results suggest that an increase in transcription of the VEGF promoter by at-RA is mediated through Sp1 site, and both new protein synthesis and tyrosine kinase activation are necessary for this induction. Because VEGF can promote neovascularization in cancer cells, an induction of VEGF by at-RA may preclude the therapeutic application of at-RA to cancer patients.

Antitumor activity of bax and p53 naked gene transfer in lung cancer: in vitro and in vivo analysis.

In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and lung tumor regression. In contrast, the therapeutic potential of bax, another apoptosis-inducing gene, has not been described. We compared p53 and bax cytotoxic effects by transient transfection of an average of 25 +/- 5% of the H-322 and H-358 bronchioloalveolar carcinoma cell lines in vitro. Under these conditions, bax expression killed 70 to 90% of the transfected cells whereas p53 killed only 40% of them. The killing activity of both genes involved apoptosis, as shown by TUNEL staining. Surprisingly, BrdU incorporation indicated that the cells that did resist Bax toxicity were blocked in the pre-S phase of the cell cycle, a result expected for p53 only. In vivo, repeated injections of naked DNA encoding Bax or p53 inhibited the growth of 4-mm preestablished H-322 tumors in nude mice. Growth retardation only, and not inhibition, was observed in H-358, a poorly transfectable and rapidly growing tumor. These results indicate that Bax and p53 share a similar, strong antitumor activity in vivo, even if the former is a more potent inducer of apoptosis in vitro.

Production of gastrin-releasing peptide by a non-small cell lung carcinoma cell line adapted to serum-free and growth factor-free conditions.

Gastrin-releasing peptide is an important growth-modulating factor in developing lung epithelium. It is known to be produced by small cell carcinomas of the lung, and an autocrine loop involving gastrin-releasing peptide and its receptor has been demonstrated in many small cell lung tumors. We investigated whether such an autocrine loop could also be demonstrated in non-small cell lung carcinoma, since gastrin-releasing peptide is known to stimulate human bronchial epithelial cells, from which non-small cell tumors should emerge. We report here that gastrin-releasing peptide is produced by a bronchiolo-alveolar carcinoma cell line (A549) adapted to serum-free and growth factor-free conditions. A549 cells adapted to these conditions, termed A549-R0 cells, display extensive membrane interdigitations, Golgi apparatus, and secretory-like granules, and grow as a mixture of attached colonies and floating cells. Gastrin-releasing peptide is present in the conditioned medium produced by A549-R0 cells. Colony formation of cells derived from a squamous cell carcinoma of the lung, 239T, was stimulated 9-fold by A549-R0 conditioned medium or by authentic gastrin-releasing peptide, measured in serum-free conditions. The growth stimulatory activity was inhibited by a monoclonal antibody to gastrin-releasing peptide. Transcripts for receptors for the bombesin family of peptides were also demonstrated in A549-R0 cells and 239T cells. These results demonstrate that non-small cell lung carcinomas can secrete gastrin-releasing peptide and can also respond to the peptide.