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Endocrinology Vol. 144, No. 8 3683-3691
Copyright © 2003 by The Endocrine Society

Adenovirus-Mediated Expression of Human Prorelaxin Promotes the Invasive Potential of Canine Mammary Cancer Cells

Josh D. Silvertown, Brad J. Geddes and Alastair J. S. Summerlee

Department of Biomedical Sciences (J.D.S., A.J.S.S.), Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada; and Millennium Pharmaceuticals Inc. (B.J.G.), Cambridge, Massachusetts 02139

Address all correspondence and requests for reprints to: Dr. Alastair J. S. Summerlee, Office of the Provost and Vice President (Academic), University of Guelph, University Centre, Fourth Floor, Guelph, Ontario, N1G 2W1, Canada. E-mail: a.summerlee{at}exec.uoguelph.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ENDOCRINE SYSTEM plays a complex role in the physiology of cellular movement during processes such as fetal development, mammary gland involution, and metastasis (1). In the female, the relaxin peptide is a factor associated with the physiological development and neoplastic growth of the human mammary gland (2, 3). Indeed, the human-1, human-2, and relaxin-like factor (or INSL3) peptides have been shown to be up-regulated in human neoplastic mammary tissues (2, 4). A number of studies suggesting the potential roles of relaxin in cancer have reported this peptide to be involved in tumor cell growth and differentiation (5, 6, 7), the promotion of tumor neovascularization and angiogenesis via the possible up-regulation of nitric oxide synthase (8, 9), and vascular endothelial growth factor (10, 11). Moreover, relaxin regulates the complex interactions of the plasminogen activator (12, 13) and matrix metalloproteinases (MMP)/tissue inhibitors of MMP systems (14, 15, 16, 17, 18) on the extracellular matrix, to facilitate tumor cell attachment, migration, and invasion (18). Each of these factors plays a significant role in transforming benign tumors into the malignant state; and relaxin, therefore, has the potential to affect key pro- and antiinvasive stimuli in many tissue and cell types in mammals.

The recent discovery of two relaxin receptors, LGR7 and LGR8, as members of the family of leucine-rich repeat- containing G protein-coupled receptors (LGR) (19) opens the field for a greater understanding of the mechanistic actions of the relaxin family in signal cascades and cellular signaling, specifically for tumor progression. Moreover, relaxin-like factor, or INSL3, is another ligand of the LGR8 but not the LGR7 receptor (20). This is further supported by the serendipitous discovery that the LGR8 receptor possesses high homology with the mouse Great gene, and male Great-/- mice display a cryptorchid phenotype similar to that in INSL3-deficient mice (21, 22, 23). The characterization of the relaxin LGR7/8 receptors has only been explored in human cell lines (19) and in murine (20), rodent (24), and equine tissues (25).

The relaxin peptide is established as a reproductive hormone involved in mammary gland development during lactation and in preparing the birth canal for passage of the fetus during parturition, by ripening the cervix and softening the uterine tissues (26). In recent years, the role of relaxin has expanded to include, enhancing coronary (27, 28) and renal (29, 30) function, aiding blastocyst implantation by promoting endometrial ripening (31), and stimulating behavioral changes in drinking via the central delivery of the peptide (32). We have developed a tool for further evaluating the pleiotropic actions of relaxin in mammals. We describe the first report of the development and preliminary characterization of an adenoviral vector expressing human preprorelaxin (hH2).

Replication-defective recombinant adenoviral (rAd) vectors have been employed as a highly efficient transgene delivery system (33). One of the advantages of this vector is its ability to infect a wide variety of cell types both in vitro and in vivo (33). Control of transgene transcription by regulating the promoter is a key feature of this vector, providing a mechanism for the on/off regulation of the transgene expression. One of the most prominent regulatable transcription systems is that of the tetracycline-responsive promoters designed by Gossen and Bujard (34) in 1992. Our vector makes use of a tetracycline-responsive element (TRE), which controls the bicistronic expression of the H2 cDNA and enhanced green fluorescent protein (EGFP) reporter gene via an independent ribosomal entry site (IRES). We present data indicating that controlled production of prorelaxin from this adenovirus is sufficient to facilitate the in vitro migration of canine mammary cancer cells through a laminin-coated porous membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and medium
Human embryonic kidney 293 (HEK-293) cell line (gift from Dr. Alan Wildeman, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada); human mammary cancer cell line MDA-MB-435 (gift from Dr. Kelly Meckling-Gill, Department of Human Biology and Nutrition, University of Guelph); and a canine mammary cancer cell line, CF33.MT (ATCC, Manassas, VA) were each grown in DMEM (catalog no. 12800–017; Life Technologies, Inc., Burlington, Ontario), supplemented with 10% fetal bovine serum (FBS; Life Technologies, Inc.). Cells of the human monocytic THP-1 cell line (ATCC) were grown in RPMI 1640 medium (ATCC) with 2 mM L-glutamine, adjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, and supplemented with 0.05 mM 2-mercaptoethanol and 10% FBS. All cell lines were maintained at 37 C in 5% C02.

Construction of adenoviral shuttle plasmid
Shuttle plasmids were constructed using standard protocols (35). ElectroMAX DH10B Escherichia coli cells (Life Technologies, Inc.) were transformed by electroporation. Plasmid isolation was prepared using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA). A 612-bp human H2 cDNA fragment was isolated from the pMAL-p2x-hRLXH2 plasmid (gift from Howard Florey Institute, University of Melbourne, Australia) and subcloned into the multiple cloning site of an IRES-containing adenovirus shuttle plasmid, pLEII{phi}-IRES-EGFP (36), to create pLEII{phi}-H2-IRES-EGFP. This plasmid contains an expression cassette of approximately 2.8 kb in which a tet-regulatable promoter drives the simultaneous expression of both the H2 transgene and an EGFP (37) reporter gene via an IRES element (38). Viral plasmid constructs were screened by HindIII (New England Biolabs, Inc., Mississauga, Ontario, Canada) digestion and unique band identification on 1% agarose gel electrophoresis, followed by DNA sequencing using PE Applied Biosystems (Foster City, CA) automated sequencer located at the University of Guelph Molecular Supercentre.

Rescue of Ad-H2-IRES-EGFP vectors
rAd constructs were generated by cotransfection of adenovirus shuttle plasmids with a plasmid containing the adenoviral backbone DNA according to established techniques (39). To enhance transfection results, pLEII{phi}-H2-IRES-EGFP and pLEII{phi}-IRES-EGFP DNA were prepared using endotoxin-free maxi preparation kits (Endo-Free Maxi Prep Kit; QIAGEN). To facilitate homologous recombination on transfection, the shuttle plasmid was linearized with SwaI (New England Biolabs, Inc.). Cotransfection with 4 µg shuttle plasmid, pLEII{phi}-H2-IRES-EGFP, and 0.5 µg of the helper plasmid containing the adenovirus backbone (QBI Viral DNA; Qbiogene, Inc., Carlsbad, CA) in the E1a transcomplementing cell line, HEK-293, was achieved with Lipofectamine Plus (Life Technologies, Inc.), according to the manufacturer’s instructions. Construction of a rAd using a control shuttle vector, pLEII{phi}-IRES-EGFP (without H2 cDNA) was done in parallel. After 6 h of transfection, the transfection medium was replaced with an agar overlay solution [1:1 mix of 2% low-melting-point agarose from Life Technologies, Inc. and 2x DMEM from Life Technologies, Inc., supplemented with 10% FBS (Life Technologies, Inc.) and MgCl2 (Fisher, Nepean, Ontario, Canada)]. Plaques appeared by d 11 and were isolated on d 16. Individual rAd plaques were monitored with an inverted fluorescent microscope. Wild-type clones could be differentiated from the rAd because only rAd express EGFP. Plaques were obtained by double plaque purification, followed by propagation in 80% confluent HEK-293 monolayers. To isolate a viral suspension for each of the rAd vectors, culture medium from the infected HEK-293 cultures showing complete cytopathic effect was collected, freeze-thawed three times, and separated from cellular precipitate by brief centrifugation and 0.2 µm filter-sterilization. Propagation and isolation of the transactivating adenovirus (Ad-tTA) were achieved as described above. A standard plaque assay was employed to determine that the plaque-forming units/ml for each of the Ad-IRES-EGFP, Ad-H2-IRES-EGFP, and Ad-tTA vectors was 9.0 x 108, 1.1 x 109, and 2.55 x 109 plaque-forming units /ml, respectively. Viruses were used for transfection at a multiplicity of infection (MOI) of 100, unless otherwise noted. Preliminary data with Western blot analysis showed that an MOI of 100 resulted in the strongest expression of recombinant hH2 (rhH2), without compromising cell viability (data not shown). Linear construct maps for each vector are illustrated in Fig. 1Go.



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  		FIG. 1. Linear vector maps of the three rAd constructs used in this study: tetracycline transactivator vector (Ad-tTA), control vector (Ad-IRES-EGFP), and H2-expressing vector (Ad-H2-IRES-EGFP). ITR, Inverted terminal repeat; CMV, cytomegalovirus promoter; Poly(A), polyadenylation site; PCMV, minimal CMV promoter; tetR, tetracycline repressor; VP16, activating domain of virion protein 16 of herpes simplex virus.

 
Identification of candidate vectors
To determine which rAd clone optimally expressed rhH2, Western blotting was performed. Briefly, an ON/OFF assay that exploits the tetracycline-regulatable promoter of the Ad-H2-IRES-EGFP vectors was conducted. Two nontranscomplementing mammary cancer cell lines were used: MDA-MB-435 (human) and CF33.Mt (canine). Cell cultures were transfected either with Ad-H2-IRES-EGFP (OFF), or both Ad-tTA and Ad-H2-IRES-EGFP (ON). Samples of culture media were collected, suspended in 30% acetonitrile, lyophilized, and concentrated 10-fold. The concentrated conditioned media from each of the ON and OFF samples were run on a 12% polyacrylamide gel under reducing conditions and transferred to Immobilon-P PVDF membranes (Millipore Corp., Bedford, MA). Colorimetric detection with rabbit antiporcine relaxin antiserum (1:1000; gift from Dr. O. D. Sherwood, University of Illinois, Urbana, IL) and HRP antigoat-IgG conjugate antibody (Immun-Blot; Bio-Rad Laboratories, Inc., Toronto, Ontario, Canada) identified the rAd clones, which successfully produced the rhH2 peptide. To confirm the findings of the Western blotting, a rapid method for the isolation of viral DNA was conducted (40), followed by diagnostic evaluation with HindIII (New England Biolabs, Inc.) digests run on 1% agarose gel electrophoresis (data not shown).

Biological mass spectrometry (MS)
Samples of concentrated conditioned medium were run on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue G-250 (Bio-Rad Laboratories, Inc.). The unique band, present only in the sample coinfected with Ad-H2-IRES-EGFP and Ad-tTA (ON), was excised for MS, using a Bruker Reflex III matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) instrument (Department of Molecular Biology and Genetics, University of Guelph). Analysis of the peptide samples by MS was performed in quadruplicate. Briefly, the protein was destained, eluted from the gel, and subjected to sequencing grade trypsin (Roche Molecular Biochemicals, Laval, Quebec, Canada) digestion, and desalted using C18 ZipTips (Millipore Corp.). Samples were applied to a MALDI-TOF target plate. MS analysis was performed using a Reflex III MALDI-TOF instrument (Bruker) equipped with a nitrogen laser (337 nm) operating in reflectron mode. All MALDI-TOF spectra were internally calibrated using adrenocorticotropic hormone human fragment 18–39 (Sigma, Oakville, Ontario, Canada). Peaks were assigned using the XTOF software (Bruker). The ExPASy proteomics server (Swiss Institute of Bioinformatics, Basel, Switzerland) was employed to provide the theoretical protein fragments resulting from trypsin cleavage of prorelaxin. These predicted values were compared with actual protein samples using less than a 0.1% error margin. The ProFound (http://prowl.rockefeller.edu/PROWL/prowl.html; Rockefeller University, New York, NY) database search engine was used to identify the protein by comparing the masses of peptides from the proteolytic digestion to the masses obtained from a theoretical digest of proteins within the National Center for Biotechnology Information (NCBI) nonredundant protein database. The ranking of the candidate proteins was based on their calculated probability.

Quantitation of relaxin transgene expression
To determine the approximate amount of rhH2 peptide released in the medium and retained in the cell, a rhRLX ELISA (reagents were a gift from Dr. Elaine Unemori, Connetics, Palo Alto, CA) was conducted as described in Parsell et al. (41) (1996). Approximately 600 µl conditioned medium from cultures of MDA-MB-435 and CF33.Mt cells, cotransfected with Ad-tTA and with either Ad-IRES-EGFP or Ad-H2-IRES-EGFP in Lab-Tek two-well Chamber Slides (Nunc, Inc., Naperville, IL), was analyzed for rhH2 levels. To collect cellular lysate, cells were washed in PBS, suspended in 100 µl cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl2, 0.5 mM MgCl2, 0.2 mM EGTA, 1% Triton X-100, protease inhibitors, aprotinin and phenylmethylsulfonylfluoride, were added at final concentrations of 20 µg/ml and 1 mM, respectively; Sigma] and placed on ice for 60 min, and supernatant was collected after centrifugation at 13,000 rpm for 20 min at 4 C.

Bioactivity of adenovirally expressed rhH2
Two cell-based systems were employed to evaluate the biological activity of the rhH2 produced from the Ad-H2-IRES-EGFP vector. First, the ability of the Ad-H2-IRES-EGFP vector to induce cAMP production was tested in the established THP-1 cAMP bioassay (41, 42). Briefly, THP-1 cells were seeded at a density of 8 x 104 viable cells per well of a 96-well plate. Cells were allowed to equilibrate at 37 C in a 5% C02 incubator for 2 h. Cells were subjected to d-2 concentrated conditioned culture medium (10x) of vector-infected CF33.Mt cell cultures for 30 min, at final concentrations of approximately 0.2 and 0.4 ng/ml, determined by the rhH2 ELISA. Equal volumes of concentrated conditioned medium from OFF samples were prepared and added to the THP-1 cell cultures in parallel. Samples were diluted in assay diluent (THP-1 culture medium, 0.1% BSA, 0.01% polysorbate 80; Sigma), containing forskolin (Sigma) and isobutylmethylxanthine (IBMX; Sigma) at final concentrations of 1 µM and 50 µM, respectively. Total levels of cAMP (intracellular and extracellular) were determined with the cAMP Biotrak E1A Assay (Amersham Biosciences Corporation, Piscataway, NJ) according to the manufacturer’s instructions. All samples were assayed in triplicate.

The second bioassay employed an in vitro cell migration model (43). Briefly, 1 µg laminin, diluted in 29 µl sterile water (Sigma), was coated on 8.0-µm porous polycarbonate membranes of a Transwell chamber (Costar, Fisher) and allowed to dry in a sterile laminar flow hood. CF33.Mt cells were seeded at a density of 1 x 104 cells per Transwell chamber in culture medium supplemented with 1% FBS and treated with varying concentrations of rhH2 (Howard Florey Institute) ranging from 0–250 ng/ml. At 24 h, Transwell chambers were washed in chilled PBS, fixed in a 1:1 solution of PBS and 100% methanol for 5 min, followed by a 10-min incubation in 100% methanol. Nonmigrated cells were mechanically removed with cotton-tips (44). Migrated cells were stained in a 0.4% Giemsa stain (Sigma) solution for 15 min. Using a light microscope under a x40 magnification, cell numbers in five discrete fields of equal area on each membrane were counted. A central field and four outer, nonoverlapping fields were selected. The counts were averaged and analyzed for statistical significance.

The effect of adenovirus-mediated prorelaxin expression on CF33.Mt cell migration was also investigated. CF33.Mt cells, transfected with Ad-H2-IRES-EGFP or Ad-IRES-EGFP in the presence or absence of Ad-tTA, were harvested on d 2 and seeded and processed as described above. To investigate further the effects of viral vectors on cell migration, CF33.Mt cells were coinfected with increasing MOIs, ranging from 0–250, for each the Ad-H2-IRES-EGFP and Ad-tTA vectors.

Cell proliferation assay
To confirm that the effect on CF33.Mt cell migration was not attributable to cell proliferation, Ad-H2-IRES-EGFP- and Ad-IRES-EGFP- infected cells, with or without Ad-tTA, were harvested on d 2 of Ad-infection and seeded at a density of 3.5 x 103 cells per well in a 96-well dish. To determine whether the presence of laminin may have had an effect on cellular proliferation, experiments were carried out using laminin-coated and uncoated culture vessels. Culture vessels were coated with laminin as described above. The amount of laminin used to coat the 96-well culture vessel was modified to account for the difference in surface area, compared with the Transwell chamber. At 24 h, cell proliferation was measured with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI) and read at 490 nm on an ELX-800 Bio-Tek (Winooski, VT) Microplate reader. Relative absorbencies at 490 nm were normalized as a percent of the Ad-IRES-EGFP (OFF) samples. Preliminary studies were conducted to eliminate the possibility that the EGFP fluorescence may interfere with the cell proliferation absorbance readings. Although, the EGFP chromophore has optimal excitation and emission wavelengths of 488 and 509 nm (45), respectively, the interference of this fluorescence was not a factor in the readings. These studies determined negligible differences in fluorescence at 490-nm Ad-infected CF33.Mt cultures at 24 and 48 h. This assay was also carried out for the effects of rhH2 (250 ng/ml) on CF33.MT cells at 24 h.

Statistical analysis
Samples were performed in triplicate and experiments repeated three times. By employing Statistical Analysis System (SAS) software, all data were expressed as means with ± SEM (46). A P value of less than {alpha} = 0.05 was considered to be statistically significant. General linear mixed models using SAS procedures "mixed" and "glm" were employed. Data (see Fig. 6Go, A–C) were analyzed using a random complete block design split-plot with subsampling and tested with a modified Tukey test (see Fig. 6AGo) and a one-sided Dunnet’s test (see Fig. 6BGo), and Student’s t test (see Fig. 6CGo) was conducted as a follow-up to an ANOVA on the indicated pair only (47).



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  		FIG. 6. CF33.Mt 24-h cell migration on laminin-coated, 8-µm porous membranes using in vitro Transwell cell migration assays. To ensure that appropriate comparisons were made between count fields (within and between experiments), statistical analysis was carried out using three tests (see Materials and Methods; W. Sears, Statistical Consultant, Ontario Veterinary College, University of Guelph). A, Cell migration with increasing concentrations of rhH2 (*, P < 0.05, compared with controls, by a modified Tukey test). B, Ad-infected CF33.Mt cell migration with Ad-IRES-EGFP or Ad-H2-IRES-EGFP (MOI 100) with or without Ad-tTA (MOI 100) (*, P < 0.05, compared with indicated transfected controls, analyzed using a one-sided Dunnet’s test). C, Ad-infected CF33.Mt cell migration samples with varying multiplicities of coinfection for each of the Ad-H2-IRES-EGFP and Ad-tTA vectors (*, P < 0.05, compared with uninfected controls, analyzed using a Student’s t test as a follow-up to an ANOVA on the indicated pair only). Bars, Mean ± SEM; n = 3.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of E1-deleted regulatable adenoviral vectors expressing human relaxin
An E1-deleted adenoviral vector with a tetracycline- responsive promoter driving the bicistronic transcription of H2 and EGFP transgenes was constructed. An important feature of this vector is the regulatability of the transgenes by the transactivating vector, Ad-tTA. Recombinant vectors were initially identified under a fluorescent microscope that exploited the EGFP reporter protein. To confirm that the candidate vectors were indeed recombinant products vs. wild-type, HindIII restriction enzyme analysis was performed on viral DNA. A unique band of approximately 887 bp was observed from Ad-H2-IRES-EGFP vectors vs. the control Ad-IRES-EGFP vectors, which do not contain this transgene (data not shown). Western blots were also conducted on concentrated conditioned media from cells transfected with Ad-H2-IRES-EGFP in the presence or absence of Ad-tTA. To observe an immunoreactive band with the colorimetric Western blotting system, d-2 conditioned media of infected MDA-MB-435 and CF33.Mt cell cultures were concentrated 10-fold. Fig. 2Go illustrates the immunodetection of the CF33.Mt ON/OFF samples, where one band with an approximate size of 18 kDa was observed. This band was only observed in the ON sample containing the Ad-H2-IRES-EGFP; no immunoreactive bands were observed in the ON samples with the Ad-IRES-EGFP vector. Tryptic digestion has been established as a verification tool for relaxin identification (48). In the present study, 26 monoisotopic masses derived from the MALDI-TOF spectrum of proteolytic products were submitted to the ProFound search engine, which confirmed that the peptides resulting from tryptic digestion matched a single protein: human H2 prorelaxin. The normalized probability of the identified human H2 prorelaxin protein was 1. The probabilities of the next 2 ranked candidates were 4.5 x 10-11 and 1.1 x 10-11, respectively.



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  		FIG. 2. A colorimetric Western blot of d-2 concentrated conditioned medium from transfected CF33.Mt cell cultures. OFF samples: lanes 1 (Ad-IRES-EGFP) and 3 (Ad-H2-IRES-EGFP). ON samples: lanes 2 (Ad-IRES-EGFP and Ad-tTA), and 4 (Ad-H2-IRES-EGFP and Ad-tTA). Lanes 5–7 contain 1.5, 3, and 4.5 µg recombinant human relaxin, respectively. Lane 8 contains conditioned medium from uninfected CF33.Mt cell cultures.

 
Adenovirus-mediated prorelaxin expression
Candidate rAd vectors for each construct were selected based on three variables: 1) confirmation of integration of the H2 cDNA in the rAd backbone with HindIII digestion of viral DNA; 2) a relaxin-like protein in a Western blot; and 3) precise promoter control of transgene expression with Ad-tTA in the ON/OFF assay in two mammary cancer cell types. Regulation of the tetracycline promoter with the transactivating virus (Ad-tTA) is illustrated in Fig. 3Go. Green fluorescence is observed only when cells are infected with either the Ad-IRES-EGFP or Ad-H2-IRES-EGFP, and Ad-tTA.



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  		FIG. 3. Regulation of Ad-H2-IRES-EGFP with Ad-tTA in CF33.Mt cell cultures visualized with an inverted fluorescent microscope. A, ON: CF33.Mt cultures cotransfected with Ad-H2-IRES-EGFP and Ad-tTA. B, OFF: CF33.Mt cultures transfected with the Ad-H2-IRES-EGFP vector.

 
Conditioned culture medium from the ON/OFF assays was measured for relaxin, using an established rhRLX ELISA (Fig. 4Go). Both the canine CF33.Mt and human MDA-MB-435 mammary cancer cell lines were found to secrete relatively higher levels of rhH2 protein, compared with retained protein in the cell (data not shown). Both cell lines were used to characterize and compare the differences of adenoviral- mediated rhH2 expression and posttranscriptional processing. The approximate levels of rhH2 were quantified at three time points for the CF33.Mt and MDA-MB-435 cell lines. The rhH2 levels were optimally secreted on d 2 for the CF33.Mt cell line, with 3600 ± 41.7 pg/1 x 106 cells/ml; compared with d 4 and 6, with approximate H2 levels of 2200 ± 20.1 and 2700 ± 13.2 pg/1 x 106 cells/ml, respectively. The rhH2 levels were optimally secreted on d 4 for the MDA-MB-435 cell line, with 8315 ± 102.4 pg/1 x 106 cells/ml; compared with d 2 and 6, with approximate H2 levels of 5175 ± 75.75 and 2990 ± 28.7 pg/1 x 106 cells/ml, respectively. A comparison of the additive prorelaxin production for CF33.Mt and MDA-MB-435 cells over 6 d is shown in Fig. 4Go.



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  		FIG. 4. Adenoviral-mediated secreted levels of rhH2 over a 6-d period, as detected with an rhH2 ELISA. CF33.Mt and MDA-MB-435 cell cultures were cotransfected with Ad-H2-IRES-EGFP and Ad-tTA. n = 3.

 
THP-1 cAMP ELISA
The bioactivity of the relaxin expressed from this viral vector was confirmed after secreted prorelaxin forms in CF33.Mt cell culture medium stimulated cAMP production in THP-1 cells (Fig. 5Go). The rhH2 levels of 0.2 and 0.4 ng/ml promoted the up-regulation of total cAMP (intra- and extracellular) with levels of approximately 2.8 ± 0.16 and 4.1 ± 0.2 pmol/well, respectively. The total cAMP levels, measured after treatment with equal volumes of samples from the OFF cell cultures (where rhH2 was not detected in the rhH2 ELISA), exhibited far lower levels of 0.66 ± 0.03 pmol and 0.62 ± 0.02 pmol/well, respectively.



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  		FIG. 5. The production of total cAMP from THP-1 cells after a 30-min incubation with volumes of conditioned medium of Ad-H2-IRES-EGFP- and Ad-tTA-coinfected CF33.Mt cell cultures containing 0.2 and 0.4 ng/ml rhH2, supplemented with 1 µM and 50 µM forskolin and IBMX, respectively. Total levels of cAMP (pmol/well) was determined with the cAMP Biotrak E1A Assay (Amersham Biosciences Corporation). Bars, Mean ± SEM; *, P < 0.05, compared with the Ad-EGFP control (ON; 0.2 and 0.4 ng/ml); n = 3.

 
Transwell cell migration assay
The incubation of increasing levels of rhH2 to CF33.Mt cells seeded in the in vitro Transwell migration assay stimulated an increase in cellular migration at levels of 250 ng/ml (146 ± 14 cells/field), compared with saline-treated controls (95 ± 14 cells/field, Fig. 6AGo, P = 0.0358), and cells incubated with 100 ng/ml (94.95 ± 14 cells/field, P = 0.0341) and 150 ng/ml (98.08 ± 14 cells/field, P = 0.0439) of rhH2, respectively. Unlike the responses seen on laminin-coated wells, cells seeded on uncoated Transwell membranes did not exhibit a significant migratory response to relaxin or saline treatment (P > 0.05).

Cotransfection of CF33.Mt cells with Ad-H2-IRES-EGFP and Ad-tTA (ON), but not Ad-IRES-EGFP and Ad-tTA (ON), resulted in increased migration (Fig. 6BGo). Transfection of cells with either Ad-IRES-EGFP or Ad-H2-IRES-EGFP alone (OFF) did not result in increased migration. After 24 h, cells cotransfected with Ad-H2-IRES-EGFP and Ad-tTA (ON) had a significantly greater number of migrated cells (151 ± 28 cells/field), compared with samples transduced with just the Ad-H2-IRES-EGFP (OFF, 111 ± 28 cells/field, P = 0.015), Ad-IRES-EGFP (ON, 98 ± 28 cells/field, P = 0.002), or Ad-IRES-EGFP (OFF, 98 ± 28 cells/field, P = 0.002).

The increasing number of viral particles present in the cells had a dose-dependent effect on inhibiting cellular migration (Fig. 6CGo). Although an MOI of 10 caused a significant increase in migratory capacity (113 ± 16 cells/field), compared with saline-treated samples (64 ± 8 cells/field, P < 0.05), vector MOIs greater than 10 resulted in a significant inhibition of cellular migration. It is important to note that, because cells were transfected with two vectors (Ad-H2-IRES-EGFP and Ad-tTA), each at the indicated MOI values, actual MOIs are doubled.

Cell proliferation assay
To ensure that the enhanced cell migration observed was not attributable to a mitogenic effect of relaxin, the possible proliferative effects of Ad-mediated prorelaxin expression and exogenous rhH2 delivery were determined. The number of viable cells infected with the Ad constructs was determined at 24 h. Ad-H2-IRES-EGFP (ON) samples, which were found to have an enhanced migratory capacity, exhibited negligible differences in percentage of cell growth over 24 h, compared with the Ad-H2-IRES-EGFP (OFF) or Ad-IRES-EGFP (ON/OFF) samples (Fig. 7Go). Similarly, there were no differences in cellular proliferation after 24 h of incubation with exogenous relaxin at 250 ng/ml in CF33.Mt cultures, compared with saline-treated controls. Moreover, the presence of laminin on the substratum of the cell culture vessels did not affect cell proliferation or provide an enhanced prolific nature to any of the transfected cell combinations: Ad-IRES-EGFP (ON/OFF) or Ad-H2-IRES-EGFP (ON/OFF).



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  		FIG. 7. Ad-infected CF33.Mt cellular proliferation at 24 h with Ad-IRES-EGFP or Ad-H2-IRES-EGFP with or without Ad-tTA. Proliferation was measured on culture vessels coated with or without laminin. Relative absorbencies are normalized as a percent of the CF33.Mt cells transfected with Ad-IRES-EGFP (OFF; n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study is the first to report the characterization of an adenoviral vector expressing the human H2 relaxin cDNA. Vector-mediated expression of the H2 cDNA in the CF33.Mt cell line resulted in production of prorelaxin, which was confirmed in immunoblot and MALDI-TOF analyses. High levels of adenoviral-mediated prorelaxin secreted by two mammary cancer cell lines were quantitated in a direct sandwich ELISA, although there were quantitative differences between the two cell lines. Bioactivity of the rhH2 expressed by the vector was demonstrated in the established THP-1 cAMP ELISA bioassay and confirmed in an in vitro Transwell cell migration model. Both adenoviral and exogenous delivery of rhH2 to canine mammary cancer cells, seeded on laminin-coated porous membranes of a Transwell chamber, facilitated cell migration at 24 h, compared with controls, suggesting that relaxin may play a role in cell migration and invasion.

The presence of a relaxin-like protein produced by the transfected cells was confirmed by Western blot analysis. Both the canine and human cancer mammary cell cultures infected with the adenoviral vector expressing the H2 cDNA secreted prorelaxin. It is presumed that the processing of hH2 (21 kDa) to prorelaxin (18.3 kDa) involves removal of the signal peptide catalyzed by a signal peptidase in the endoplasmic reticulum (49, 50). Similar to insulin, the removal of the connecting C peptide converts prorelaxin to the mature, heterodimeric relaxin hormone, 6.5–7kDa, depending on where the C peptide is cleaved (51). This process is thought to involve enzymatic cleavage facilitated by prohormone convertases (PCs), such as PC1 (51, 52) and furin (53). Although furin and other PCs have been identified in a number of human mammary cancer cell lines and in human cancerous tissue (54, 55, 56), PCs have yet to be investigated in the MDA-MB-435 cell line; evidence for PCs in canine mammary tissues also remains to be examined. Therefore, the absence of PCs from the cell lines used in the current study may explain why unprocessed prorelaxin was detected. Similar to the present study, Reddy et al. (57) (1992), Vu et al. (53) (1993), and Zarreh-Hoshyari-Khah et al. (58) (2001) noted that the processing of prorelaxin is not essential for bioactivity, where prorelaxin was able to stimulate cAMP secretion from human endometrial cells and the human monocytic THP-1 cell line. The authors of the latter paper also propose that relaxin may be, in part, secreted via the constitutive pathway, where regulation occurs mainly at the transcriptional level vs. the translational or posttranslational levels. Therefore, in cases where relaxin is up-regulated, such as in vector-transduced cancer cells, elevated levels of relaxin precursor relative to the processed heterodimer could be secreted. Relaxin precursors are reported to be naturally occurring in the mammal. Pre- and prorelaxin-like forms are present in squamous differentiated rabbit tracheobronchial epithelial cells (59). Two immunoreactive bands, of 18 and 16.5 kDa, from rat ovary extracts during midpregnancy were postulated to be prorelaxin isoforms (60). Moreover, prorelaxin was measured in the sera, seminal plasma, and urine of human males (61).

The human mammary cancer cell line, MDA-MB-435, produced slightly higher levels of rAd-expressed pro-rhH2 than did the canine mammary cancer cell line, CF33.Mt. MDA-MB-435 cells are more prolific in culture, compared with the CF33.Mt cells; therefore, a more metabolically active cell line might produce more protein. Secreted prorelaxin levels may have been underestimated, because the ELISA employed in our study used porcine antihuman relaxin antibodies, which may not cross-react as readily with the quaternary conformation of human prorelaxin.

The discovery that relaxin dose-dependently stimulates cAMP release from THP-1 cells led to the establishment of an in vitro bioassay for relaxin peptides (41, 42). However, several studies differed in terms of relaxin source, concentration, presence or absence of IBMX (phosphodiesterase inhibitor) and/or forskolin (adenyl cyclase inducer), incubation time with relaxin, and whether or not intracellular, extracellular, or total cAMP is measured. In the present study, the THP-1 cAMP levels in the presence of IBMX, forskolin and vector-produced rhH2, after 30 min, had a profound and dose-dependent effect on cAMP release. To date, this is the first report that human prorelaxin is biologically active. This is not surprising, considering that other studies have demonstrated porcine (53, 57) and marmoset prorelaxin (58) to have biological activity.

There is increasing evidence for an involvement of relaxin in cellular migration and invasion. We reported that rhH2 affected the cellular migration of the L6 rat myoblast cell line, using assay conditions similar to those reported in the current study (43). Binder et al. (18) (2002) demonstrated that porcine relaxin could stimulate the MCF-7 and SK-BR3 human mammary cancer cell lines after 96 h. Recently, Wyatt et al. (62) (2002) showed that bronchial epithelial cells migrated in response to porcine relaxin in a wounding assay. In the present study, the biological activity of the rhH2 produced by the rAd was further tested in an established in vitro cell migration assay, using the CF33.Mt cell line. Although intraassay variation in cell migration was observed, the general trend that relaxin facilitated cellular migration was consistent. The exogenous delivery of rhH2 giving a final chamber concentration of 250 ng/ml stimulated a migratory response in CF33.Mt cells of approximately 1.5-fold, compared with levels of 150 ng/ml or less. The effect of adenovirally delivered rhH2 on CF33.Mt migration was also observed. The regulation of the transgenes (H2 and EGFP) was demonstrated in this assay, where only the samples that were coinfected with both the Ad-H2-IRES-EGFP and Ad-tTA vectors stimulated an enhanced migratory response (1.5-fold) in the CF33.Mt cells. Although exogenous relaxin levels of 250 ng/ml were required to elicit a significant difference in CF33.Mt in vitro cell migration, compared with untreated controls, it seems that far lower levels of rhH2 expressed by the Ad vector (according to the rhH2 ELISA) were required for in vitro cell migration in vector-transfected cells. It is possible that the rhH2 concentration in the immediate microenvironment of the infected cells secreting the relaxin may be at levels similar to these used in the migration assay with exogenous relaxin. Therefore, available relaxin receptors will be easily accessible for immediate binding and subsequent intracellular signaling. Moreover, we cannot discount the possibility that rhH2 could stimulate a nuclear or intracellular receptor before the relaxin is secreted from the cell. Figure 6CGo illustrates that increasing Ad vector cotransfections of and greater than MOIs of 100, suppress CF33.Mt cell migration, compared with untransfected cell samples. However, at an MOI of 10, rhH2 stimulated a significant CF33.Mt cell migratory capacity, compared with untransfected cells. Therefore, in this model, cells have a threshold for the number of viral particles they can harbor before migration is repressed. However, at lower infection levels, expression of the H2 transgene confers an enhanced migratory capacity. Taken together, it is clear that, although adenoviral transfection suppresses in vitro cellular migration, the constitutive expression of prorelaxin is secreted at levels strong enough to promote a significant effect on cellular migration.

A mechanism whereby relaxin plays a role in cellular migration may be achieved via the up-regulation of MMPs (18). Relaxin’s effect on the modulation of connective tissue in the uterus (17, 63, 64), decidua (65) and cervix (64), and in certain mammary (18) and lung (15) cells has been associated with the concurrent induction of MMPs. Therefore, it can be inferred that relaxin may confer an advantage to the treated cells by inducing the up-regulation of MMPs, which can degrade the laminin coating, a natural component of the basal lamina, thereby providing access to the porous membrane. Our laboratory is currently investigating the up- regulation of MMPs by relaxin in CF33.Mt cells.

In contrast to previously reported studies, our data do not support a mitogenic action of relaxin. Relaxin has been reported to be a mitogenic agent, stimulating DNA synthesis and cellular proliferation in cells from several species, including rat (66, 67), pig (68, 69), mouse (70), and human (5, 6). The effect of relaxin (250 ng/ml for 24 h) on CF33.Mt cell migration was not attributed to the induction of cell proliferation; this was further discounted when it was found that Ad-IRES-EGFP-infected cells (ON/OFF) did not differ in the number of viable cells after 24 h, compared with Ad-H2-IRES-EGFP-infected cells (ON/OFF). However, the culture conditions used in these experiments employed FBS levels of 1%, which may account for differences in cellular proliferation in our study. Bigazzi et al. (71) (1992) reported that, when MCF-7 cell cultures were exposed to varying levels of porcine relaxin with different fetal calf serum concentrations, varying effects in cellular proliferation were observed. It is possible that, to measure cellular proliferation with the method used in this study, greater supplementation of FBS in the culture medium is required for prorelaxin to promote significant mitogenic effects of the CF33.Mt cell line.

In the migration model employed for the current study, there are many factors that may affect the ability of relaxin to promote cellular migration: 1) relaxin concentration; 2) relaxin source; 3) cell line (i.e. tissue, species); 4) cell line passage number; 5) presence of ECM constituent(s); and 6) percentage of serum in culture chamber medium. Here, it was demonstrated that the biological activity of the relaxin Ad vector can be tested in this in vitro cell migration assay. Although intraassay variation may be observed, especially with different cell lines and their passage number, the migration assay may provide a rapid, cost-effective, noninvasive, and high-throughput semiquantitative bioassay. To the authors’ knowledge, this study is the first to use and report on the canine mammary cancer cell line, CF33.Mt. These cells were used for several reasons. First, they are sensitive to rH2 in the migration assay. Second, these cells were found to be highly susceptible to Ad infection, making it a suitable cell line to monitor the bioactivity of the rAd vector. Last, it is a mammary cancer cell line. The findings presented in the present study, contribute to the growing literature for the potential roles of relaxin in carcinogenesis. Our findings parallel a recent paper by Binder et al. (18) (2002), who reported that porcine relaxin (pRLX) induced the in vitro invasiveness of two human mammary cancer cell lines, MCF-7 and SK-BR3, after 4 d. Considering the findings presented here and those of Binder et al. (18) (2002), it is possible that relaxin may play a similar role in vivo. In support of this possibility, elevated relaxin concentrations were reported in the sera of breast cancer patients with active metastatic disease (72).

The presence of relaxin mRNA and the detection of relaxin secretion from the canine mammary gland are still not known. The dog exhibits the highest serum concentrations of any species studied to date, with circulating relaxin levels in the pregnant bitch of 8–10 µg/ml (73). Although the placenta is the main source of relaxin in the dog, relaxin present in the colostrum and the milk during lactation is maintained in hysteroovariectomized bitches, suggesting the presence of alternate relaxin sources, such as the mammary gland (74). In the present study, a rhH2 concentration of at least 250 ng/ml was required to induce a migratory response in the CF33.Mt cell line. However, in our previous study, a level of only 10 ng/ml rhH2 was needed to invoke a response in the L6 rat myoblast cell line (43). Relaxin levels reported in the pregnant rat fall in the range of 0.1–0.25 µg/ml (75). This difference could be attributed to the presence of fewer canine relaxin receptors on the CF33.Mt cells than the L6 rat cell line. It is also possible that endogenous canine relaxin is less potent, compared with the relaxins found in the human or rat, which are able to exhibit physiological activity at far lower circulating levels. Based on our findings that the canine cell line secretes rhH2 prorelaxin, it can be inferred that the peptide is stimulating an autocrine signal cascade via a putative canine relaxin receptor. There have been no published studies investigating the presence of LGR7/8 receptors in canine tissues. Furthermore, a Nucleotide BLAST search using the NCBI databases resulted in the absence of any LGR7/8 homologies with any canine protein. However, LGR7 transcripts have been detected in several canine cell lines and tissues (Klonisch, T., personal correspondence). Nevertheless, it is widely accepted, in the literature, that relaxin from various sources is bioactive across a spectrum of species. For example, human H2 and porcine relaxin have been investigated in many mammalian models, including the rat (10, 30, 32), mouse (9, 15), equine (16), guinea pig (76), goat (77), and rhesus monkey (78). Therefore, it is not surprising that human H2 relaxin and prorelaxin seem to cross-react with the canine receptor.

The adenovirus vector reported here contains a tetracycline-response element that controls the bicistronic expression of the relaxin H2 transgene and the EGFP reporter gene. The bioactivity of the adenovirus-expressed prorelaxin was demonstrated in an in vitro migration assay and validated in the established THP-1 cAMP assay (41, 42). The canine mammary cancer cell line, CF33.Mt, is responsive to both Ad-mediated and exogenous delivery of rhH2 on laminin-coated membranes of a Transwell cell migration model. Although vector regulation with antibiotics was not presented in the present study, our Ad vector exploiting the tet-off system can provide the capability to regulate expression with tetracycline or its analog, doxycycline, in vitro and in vivo (manuscript in preparation). This vector can be employed as a tool to further study the cellular, molecular, and physiological pleiotropic actions of relaxin in mammalian systems.


   Acknowledgments
 
The authors thank Dr. Jonathan Lamarre and Roman Poterski for their continual guidance and support throughout the experiments and for editing a draft of our manuscript. Gratitude is also extended to Dr. Dyanne Brewer, who conducted the MS analysis, and to William Sears, who assisted with the statistical analysis.


   Footnotes
 
This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada.

Abbreviations: EGFP, Enhanced green fluorescent protein; FBS, fetal bovine serum; HEK, human embryonic kidney; hH2, human H2 preprorelaxin; IBMX, isobutylmethylxanthine; IRES, independent ribosomal entry site; LGR, leucine-rich repeat-containing G protein-coupled receptor(s); MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MMP, matrix metalloproteinase(s); MOI, multiplicity of infection; MS, mass spectrometry; PC, prohormone convertase; rAd, recombinant adenoviral; rhH2, recombinant hH2; TRE, tetracycline responsive element.

Received February 25, 2003.

Accepted for publication April 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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