Inhibitory Effect of Adenovirus-Uteroglobin

Inhibitory effect of adenovirus-uteroglobin transduction on the growth of lung cancer cell lines Jae Cheol Lee,1,2 Kyung-Ho Park,1,2 Seon Jin Han,1,2 Chul-Gyu Yoo,1,2 Choon-Taek Lee,1,2 Sung Koo Han,1,2 Young-Soo Shim,1,2 and Young Whan Kim1,2 1Department of Internal Medicine and Lung Institute of Medical Research Center, Seoul National University College of Medicine, and 2Gene Therapy Laboratory of Clinical Research Institute, Seoul National University Hospital, 28 Youngon-Dong, Chongno-Gu, Seoul 110-744, Korea.

Uteroglobin is a secretory protein synthesized by most epithelia, including the respiratory tract. It has strong anti-inflammatory properties that appear to be related to the inhibition of phospholipase A2. Recent experimental evidence indicates that uteroglobin has an inhibitory effect on the proliferation and invasion of cancer cells. We investigated the effects of the adenovirus- uteroglobin (ad-UG) transduction on the growth of lung cancer cell lines, which did not express the uteroglobin gene. Upon transduction of ad-UG, the rate of cell growth and the ability to produce colonies in soft agar were evaluated. Cell cycle analysis, Western blot for cell cycle-related proteins and annexin V staining for apoptosis were carried out to see if they were associated with the changes in cell growth. All the tested lung cancer cell lines did not express the uteroglobin gene. The growth rates, and colony- forming ability of transformed cells, were significantly inhibited by the induction of uteroglobin gene expression. The DNA histogram showed that the cell fraction of the G2/M phase was increased, and this G2/M phase arrest was related to a decrease of cdk1 and cyclin A. However, a fraction of apoptotic cells were same as the control. From these results, uteroglobin is thought to have an inhibitory effect on the growth of lung cancer cells. This suggests a potential role for uteroglobin in gene therapy for lung cancer. Cancer Gene Therapy (2003) 10, 287–293. doi:10.1038/sj.cgt.7700569

Keywords: uteroglobin; adenovirus; lung cancer; gene therapy

teroglobin is a steroid-inducible, low molecular mass structure.6 Uteroglobin is an evolutionarily conserved U(15.8 kDa) protein, synthesized by the mucosal protein with important physiologic functions. It has epithelia ofmany organs, including those ofthe thymus, potent anti-inflammatory and immunomodulatory abil- pituitary gland, respiratory and gastrointestinal tracts, ities by its inhibition ofthe activity ofphospholipase and prostate and mammary gland.1 It is a secretory A2.7,8 It is also known to inhibit human and rabbit protein found in blood2 and urine.3 Uteroglobin was first phagocyte chemotaxis and phagocytosis.9 Mukherjee discovered in rabbit uterine fluids, as a critical element, et al10 proposed that uteroglobin plays a specific role in having a stimulatory effect on the growth of blastocysts in masking the antigenicity ofdeveloping embryos during early pregnancy, so was named blastokinin.4 Since then, implantation. From these results, it is suggested that many different names have been ascribed to this protein, uteroglobin may protect the developing embryo from based on the tissue or body fluid in which it was detected, immunologic attack on the maternal side, not only by or molecules it interacted with, such as retinol and binding to antigenic determinants ofembryonic cells, but progesterone. For this reason, it is also known as Clara also by impairing migration ofphagocytes, and thus cell secretory protein, lacryglobin, urine protein-1, retinol- phagocytosis.11 Normal bronchial epithelia express the binding protein and progesterone-binding protein.5 This UG gene at a high level. However, neither adenocarcino- confusing nomenclature led to a consensus for a new ma, nor cell lines, derived from the lung express the UG generic family name. Secretoglobin has been officially gene, or express it at a markedly reduced level.12 approved as the new generic name referring to the Interestingly, induced expression ofUG in some cancer uteroglobin-like molecules with common characteristics, cells results in the loss of their transformed phenotype, for including secretion, small size, alpha-helical and dimeric example, invasiveness, adhesiveness to fibronectin and anchorage-independent growth.13,14 Saxon et al. reported the introduction ofa single copy ofthe human chromo- Received June 27, 2002. Address correspondence and reprint requests to: Dr Young Whan some 11 that completely suppressed the tumorigenic Kim, Department of Internal Medicine, Seoul National University phenotype ofHeLa cells, and it was indicative ofthe Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea. E- presence ofthe tumor-suppressor gene(s) on this chromo- 15 mail: [email protected] some. The fact that the location of the UG gene has Adenovirus-uteroglobin transduction to lung cancer cell lines JC Lee et al 288 been mapped to 11q12.3–13.1 chromosome16 is very Soft-agar clonogenic assay interesting in the light ofthe result described above. To assess the effect of uteroglobin gene transfer on in vitro Additionally, the incidence ofcancer in UG-knockout tumorigenicity, soft-agar clonogenic assays were per- mice is known to be extremely high.5 Taken together, formed. Three lung cancer cell lines (NCI-H460, NCI- these results suggest that the loss ofUG gene expression H522 and A549) were transduced with 20 MOI ofad-UG, may be a common characteristic ofcancer cells, and UG ad-null or PBS. After incubating for 24 hours, cells were may act as a tumor suppressor. To confirm this isolated, and plated in 0.2% agarose with a 1% agarose possibility, we investigated the effects of the adenovirus- 3 underlay (5 Â 10 cells/plate). Colonies greater than uteroglobin (ad-UG) transduction on the growth oflung 125 mm were counted after 4 weeks. cancer cell lines that did not express the uteroglobin gene. Cell cycle analysis NCI-H460, NCI-H522 and A549 lung cancer cells were transduced with 20 MOI of ad-UG. After incubating for Materials and methods 48 hours, cell cycle analysis was carried out using Cell lines CycleTest plus kit protocol (Becton Dickinson, San Jose, Eight human lung cancer cell lines (NCI-H358, NCI- CA). H520, NCI-H522, NCI-H2009, NCI-H1299, NCI-H460, NCI-H596 and A549) were obtained from the American Western blot for cell cycle-related proteins Type Culture Collection (Manassas, VA) and the Korean Western blots for cdk1, cyclins A and B were performed Cell Line Bank (Seoul, Korea). using the ECL Western blotting system and protocol (Amersham). Monoclonal mouse anti-human antibodies for cdk1, cyclin A and cyclin B (Pharmingen, San Diego, Preparation of recombinant ad-UG CA) were used as primary antibodies. cDNA ofuteroglobin, kindly provided by Shelhamer JH ofNational Institutes ofHealth, Bethesda, Maryland, was Annexin V staining for apoptosis cloned into BamHI restriction sites ofthe polylinker of Analyses for apoptosis were carried out using annexin V the adenoviral shuttle vector, pACCMVpLpA. The apoptosis kits (Pharmingen) in three lung cancer cell lines pACCMVpLpA-UG and pJM17 adenoviral packaging (NCI-H460, NCI-H522 and A549). At 48 and 96 hours plasmids were cotransfected into 293 cells (human renal after transduction with 20 MOI of ad-UG, cells were embryonal cells immortalized by stable transfection with isolated by a brieftrypsin treatment. The cells were E1 ofthe adenovirus) using the calcium phosphate stained with annexin V and PI, according to the precipitation method. The transfected cells were main- manufacturer’s instructions, and sorted using a FACS tained in RPMI with 2% FBS until the onset ofthe flow cytometer (Becton Dickinson). cytopathic effect. The generated adenovirus was purified three times by plaque assays and confirmed by sequencing ofthe viral DNA and Western blot analysis. A large-scale Results stock ofadenovirus was prepared by the standard CsCl method. Recombinant adenovirus, without any therapeu- Northern blot analyses showed no expression of the tic gene (ad-null), was used as the control virus in all uteroglobin gene in lung cancer cell lines experiments. Northern blot analyses were performed to see whether the gene ofuteroglobin was expressed in lung cancer cell lines or not. Eight human lung cancer cell lines (NCI-H358, Transduction of lung cancer cell lines with ad-UG NCI-H520, NCI-H522, NCI-H2009, NCI-H1299, NCI- Lung cancer cells were transduced with 20 and 100 MOI H460, NCI-H596 and A549) were used. These showed no of ad-UG or ad-null for control virus and incubated for 1 expression ofthe uteroglobin gene in any ofthe tested hour with gentle frequent shaking and then incubated lung cancer cell lines. with complete media for the experiment. The induction of uteroglobin gene expression was confirmed by Western The expression of the uteroglobin gene was induced and Northern blot analyses in cell lysates and media. after transduction of ad-UG Northern blot analyses revealed successful induction of uteroglobin gene expression following transduction of ad- Analysis of growth inhibition on lung cancer cell lines UG in lung cancer cell lines. The amount ofinduced Three lung cancer cell lines (NCI-H460, NCI-H522 and expression was proportional to the transduced viral A549) were seeded onto six-well plates (5 Â 104/well). burden (Fig 1). Uteroglobin was also detected in cultured After incubating for 24 hours, cells were transduced with media by Western blot analysis (Fig 2). This suggests that 20 MOI of ad-UG, ad-null or phosphate-buffered saline the transformed cells synthesized uteroglobin and secreted (PBS). Triplicate wells ofeach treatment were counted it into the media. Western blot analysis ofthe brochiolo- daily for 8 days using a hemocytometer. alveolar lavarge in a normal person showed that the

Cancer Gene Therapy Adenovirus-uteroglobin transduction to lung cancer cell lines JC Lee et al 289 The growth of transformed cells by ad-UG was significantly inhibited Cell count was performed daily during the culture for 8 days to evaluate the effect of transduction with ad-UG to cell growth in three lung cancer cell lines (NCI-H460, NCI-H522 and A549). The differences of growth rate between the transformed cells by ad-UG and cells transduced with ad-null or control appeared at 2 days after transduction. Thereafter, the differences of growth rate had been increasing day by day. This revealed that transduction with ad-UG inhibited the growth of transformed cells significantly compared with the cells Figure 1 Northern blot analysis for uteroglobin in lung cancer cell untransduced or transduced with ad-null in three tested lines after transduction of ad-UG. Uteroglobin gene expression was lung cancer cell lines (Fig 4). induced successfully following transduction of ad-UG in eight lung cancer cell lines. The result of Northern blot in three lung cancer cell lines (NCI-H460: large cell carcinoma, NCI-H522: adenosquamous Colony-forming ability in soft agar was suppressed cell carcinoma, A549: adenocarcinoma) is shown. The amount of expression in cells transduced with 100 MOI of ad-UG were larger Colony count was performed at 4 weeks after transduc- than that in cells transduced with 20 MOI of ad-UG, showing that the tion with ad-UG. The colony-forming ability of the amount of induced expression was proportional to the transduced transformed cells in soft agar was markedly reduced, by viral burden. more than 80% (Fig 5).

G2/M phase arrest was caused by the induction of the uteroglobin gene expression

The induction ofuteroglobin gene expression caused G2/ M phase arrest compared to the control. The fraction of G2/M phase in transduced cells ofNCI-H460 increased to 51.58% compared to 10.99% in the control and the S phase fraction simultaneously decreased to 15.24% compared to 39.02% in the control (Fig 6). The results Figure 2 Western blot analysis in cell lysates and cultured media. in cells transduced with ad-null were not different from Uteroglobin was detected both in cell lysates and cultured media by control, showing that the fraction of G2/M phase was the Western blot analysis. This reveals that the transformed cells 9.33% and S phase fraction was 36.88%. The G2/M synthesized uteroglobin and secreted it into the cultured media. The phase arrest was also found in A549 and NCI-H522 amount of secreted proteins was also proportional to the concentra- (Fig 6). tion of the transduced virus, like Northern blot analysis. Western blot for cell cycle-related proteins showed decreased levels of cdk1 and cyclin A Western blot analyses ofthe cell cycle-related proteins associated with G2/M phase showed decreased levels of cdk1 and cyclin A in three lung cancer cell lines after ad- UG transduction (Fig 7).

Figure 3 Western blot analysis in bronchioloalveolar lavage and a lung cancer cell line (NCI-H358: bronchioloalveolar cell carcinoma). Apoptosis was not induced by the transduction of Western blot analysis in bronchioloalveolar lavage (BAL) fluids ad-UG obtaind in a normal person during bronchoscopy showed that The transduction ofad-UG did not induce apoptosis at 48 uteroglobin was constitutively synthesized and secreted at a high level in normal bronchial epithelium. In NCI-H358 lung cancer cell or 96 hours (Fig 8). There were no differences of line, only the transformed cells by ad-UG produced the uteroglobin. apoptotic cell fractions between the transformed cells and control or cells transduced with ad-null. The apoptotic cell fraction in transformed cells of NCI-H460 was 0.04% at 48 hours and 1.02% at 96 hours. It was expression ofuteroglobin gene is constitutively taking 1.94% at 48 hours and 0.56% at 96 hours in the control. place at high level in normal bronchial epithelium. This Similar results were obtained in the cells transduced with result contrasted with the Western blot analysis ofNCI- ad-null, showing 0.52% at 48 hours and 0.76% at 96 H358 showing that the expression ofuteroglobin gene was hours. Also in other cell lines (NCI-H522 and A549), only detected in the transformed cells by ad-UG (Fig 3). apoptosis was not induced by the transduction ofad-UG.

Cancer Gene Therapy Adenovirus-uteroglobin transduction to lung cancer cell lines JC Lee et al 290

Figure 5 Soft-agar clonogenic assay. After transduction with 20 MOI of ad-UG, ad-null or PBS, three lung cancer cell lines (A549, NCI- H460 and NCI-H522) were plated in 0.2% agarose with a 1% agarose underlay. Colony counts after 4 weeks showed that in vitro tumorigenecity in transformed cells was markedly reduced, by more than 80%.

patients with lung cancer. The poor prognosis, showing less than 15% of5-year survival rate forthis disease, requires new therapeutic methods, such as gene therapy.17 Gene therapy is the treatment ofdisease using genetic material as a therapeutic agent. The most common approach to cancer gene therapy is the correction of genetic aberration by the introduction ofnormal genes. 18 Gene therapy using the wild-type p53 tumor-suppressor gene and adenoviral vector is the most extensively studied in lung cancer.19 Clinical trials in inoperable lung cancer have consistently revealed that adenovirus-mediated p53 gene therapy can be performed without significant adverse effects, and show potential useful clinical responses. Although it has been shown that gene therapy may be a promising tool in managing patients with inoperable lung cancer, there are many problems to resolve, including developing more effective gene delivery systems, and discovering new therapeutic genes.20 Uteroglobin is known to be a possible candidate gene, with tumor-suppressor activity. Normal bronchial epithelium produces a large amount ofuteroglobin. However, prolonged exposure carcinogens such as tobacco smoke led to reduced uteroglobin expression. On the contrary, the overexpression ofuteroglobin made immortalized bronchial epithelial cells delay to have the phenotype of 21 Figure 4 Induction of the uteroglobin gene expression markedly cancer cells in response to carcinogens. This suggests inhibited the growth of lung cancer cells. After transduction with 20 that downregulation ofuteroglobin contributes to pul- MOI of ad-UG, cells were counted daily to evaluate the effect to the monary carcinogenesis. The other evidences suggesting cell growth. The growth of transformed cells was remarkably that uteroglobin antagonize the neoplastic phenotype suppressed compared to control or cells transduced with ad-null have been accumulating. The induction ofuteroglobin in (A549, NCI-H460 and NCI-H522). Bars, SD. a lung cancer cell line caused the reduced invasiveness of cancer cells.13 Enforced expression by transfection of uteroglobin cDNA in the endometrial adenocarcinoma Discussion cell line markedly reduced growth potential and pro- Lung cancer is the most common cause ofdeath from longed the time for completion of the cell cycle.22 The cancer in many parts ofthe world. Despite recent Western blot analyses in our study showed that normal advances in diagnosis and treatment methods, there has bronchial epithelium synthesized and secreted uteroglobin been no significant improvement in the survival rate in in high levels, but lung cancer cell lines did not. These

Cancer Gene Therapy Adenovirus-uteroglobin transduction to lung cancer cell lines JC Lee et al 291

Figure 6 Cell cycle analysis. DNA histogram in NCI-H460 revealed that the induction of uteroglobin gene expression caused G2/M phase arrest. The G2/M phase fraction in transformed cells increased to 51.58% compared to 10.99% in the control and the S phase fraction simultaneously decreased to 15.24% compared to 39.02% in the control. The results of A549 and NCI-H522 were similar to those of NCI-H460.

coincide well with many previously reported data. After development ofmulticellular organisms. Cdk complexes, transfer of the uteroglobin gene to lung cancer cell lines, composed ofa regulatory cyclin subunit and a catalytic the growth ofcancer cells was significantlyinhibited and cyclin-dependent kinase subunits, regulate the processes the tumorigenecity, in soft agar, was reduced. These in cells, through the cell cycle.23 To minimize the results convincingly support the finding that uteroglobin replication ofdamaged DNA, and control the order and acts as a tumor suppressor. timing ofcell cycle transition, several checkpoints, such as Most eukaryotic cells replicate through the cell cycle. G1/S and G2/M, are present in the cell cycle.24 The G1/S Regulation ofthe cell cycle is critical forthe normal check point is regulated by cdk4-cyclin D, cdk6-cyclin D,

Cancer Gene Therapy Adenovirus-uteroglobin transduction to lung cancer cell lines JC Lee et al 292 cdk2-cyclin E and cdk2-cyclin A. The regulation Apoptosis is a physiologic mode ofcell death, which is ofthe G2/M check point is associated with cdk1- different from the accidental cell death process known as cyclin B and cdk1-cyclin A.25 Checkpoints arrest the cell necrosis. In response to extensive DNA damage, tumor- cycle providing time for repair, and induce transcription suppressor genes, like p53, induce apoptosis to prevent ofthe genes that facilitaterepair ofdamaged DNA. accumulation ofmultiple mutations that might result in Checkpoint loss causes genomic instability and has been the development ofcancer cells. 27 Methods ofcancer implicated in the evolution ofnormal cells into cancer treatment, such as chemotherapy or radiotherapy, cause cells.26 Many effectors of tumor-suppressor genes, such as damage to DNA in the proliferating cells, resulting in cell p53, function by controlling the cell cycle. In this study, death via the induction ofapoptosis. 20 Although uter- the induction ofuteroglobin expression in lung cancer oglobin seems to have many common characteristics of cells resulted in G2/M phase arrest, which was related to tumor suppressors like p53, it did not induce apoptosis. the decreased levels ofcdk1 and cyclin A. This suggests Further investigation is required to clarify how uteroglo- that uteroglobin has a functional mechanism similar to bin induces cell death through the arrest ofthe cell cycle. other tumor suppressors in the inhibition ofcancer cell Induction ofthe uteroglobin gene expression have the replication. inhibitory effect on the growth of lung cancer cell lines via the control ofcell cycle, leading to the conclusion that the uteroglobin gene is a kind oftumor-suppressor gene, so might be useful in lung cancer gene therapy.

Acknowledgments

This study was supported by a grant no. 04-1999-0140 from the Seoul National University Research Fund.

References

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Figure 8 The transduction of ad-UG did not induce apoptosis at 48 or 96 hours. FACS analysis of apoptosis was performed at 48 and 96 hours by Annexin V-FITC Apoptosis Detection Kit after transduction with 20 MOI of ad-UG. Right lower quadrant represents early apoptotic cell fraction. There were no differences of apoptotic cell fractions between the transformed cells and control or cells transduced with ad-null.

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