Activating transcription factor-1 (ATF-1) and cAMP-responsive element (CRE)-binding protein (CREB) have been implicated in cAMP and Ca2+-induced transcriptional activation. The expression of the transcription factors CREB and ATF-1 is upregulated in metastatic melanoma cells. However, how overexpression of ATF-1/CREB contributes to the acquisition of the metastatic phenotype remains unclear. Here, the effect of disrupting ATF-1 activity was investigated using intracellular expression of an inhibitory anti-ATF-1 single chain antibody fragment (ScFv). Intracellular expression of ScFv anti-ATF-1 in MeWo melanoma cells caused significant reduction in CRE-dependent promoter activation. In addition, expression of ScFv anti-ATF-1 in melanoma cells suppressed their tumorigenicity and metastatic potential in nude mice. ScFv anti-ATF-1 rendered the melanoma cells susceptible to thapsigargin-induced apoptosis in vitro and caused massive apoptosis in tumors transplanted subcutaneously into nude mice, suggesting that ATF-1 and its associated proteins act as survival factor for human melanoma cells. This is the first report to demonstrate the potential of ScFv anti-ATF-1 as an inhibitor of tumor growth and metastasis of solid tumor in vivo.
The molecular basis of human malignant melanoma progression has remained largely unknown, despite the fact that the worldwide incidence of melanoma is increasing more than any other neoplastic disease (Kopf et al., 1995). The development of malignant melanoma in humans progresses through a multistage process. The switches from melanocyte to nevi, to radial growth phase (RGP), and subsequently to vertical growth phase (VGP, metastatic phenotype) are associated with decreased dependence on growth factors, diminished anchorage dependence, and reduced contact inhibition (Fidler, 1990; Lu and Kerbel, 1994).
A large body of data concerning the molecular control of melanoma progression has come from studies using mitogens. In culture, melanocytes synergistically respond to a number of growth factors, which in contribution with each other or with 12-0-tetradecanoylphorbol-13-acetate or cAMP stimulate not only proliferation but also pigmentation (Halaban, 1993). These growth factors include several fibroblast growth factors, hepatocyte growth factor, and stem cell factor (also known as KIT ligand, MGF and steel factor), all of which stimulate tyrosine kinase receptors. As melanocyte proliferation and differentiation are positively regulated by agents that increase cAMP (Halaban et al., 1983, 1984, 1987; Huang et al., 1996), we have focused on the transcription factors ATF-1 (Activating transcription factor 1) and CREB (cAMP-responsive element-binding protein), which are known to be activated by cAMP, as possible mediators of tumor growth and metastasis of human melanoma.
ATF-1 and CREB are members of the large bZIP superfamily of transcription factors. Members of the CREB/ATF family bind to cAMP-responsive elements (CREs) within the promoter and enhancer sequences of many genes. ATF-1, CREB, and the cAMP-responsive element modulator protein (CREM) constitute the CREB/ATF subfamily within the bZIP superfamily, whose members are defined by their ability to heterodimerize with each other but not with members of other subfamilies (Meyer and Habener, 1993). ATF-1, CREB, and CREM have similar structures and are highly homologous at the amino acid sequence level, especially within the bZIP region. Despite these similarities, members of the CREB multigene subfamily have distinct biological activities.
ATF-1, CREB, and CREM may act as either positive or negative regulators of transcription. Alternative mRNA splicing produces numerous isoforms of ATF-1 and CREB, that can account for the variability in transcriptional regulatory activities (Lee and Masson, 1993; Lemaigre et al., 1993). In addition, each protein or isoform also possesses differing patterns of phosphorylation, and the specific patterns contribute to their ability as regulators of transcription (Meyer and Habener, 1993). CREB and CREM have been shown to play important roles in basal and hormone-regulated transcription and differentiation while the role of ATF-1 is less well defined. ATF-1 homodimers appear to be weaker transcriptional activators than either CREB or certain forms of CREM, since ATF-1-mediated activation is enhanced by heterodimerization with either CREB or CREM (Lee and Masson, 1993). In addition, it has been demonstrated that CREB can efficiently form heterodimers with ATF-1 rather than from the CREB homodimer (Hurst et al., 1991; Rehfuss et al., 1991).
Previous studies have demonstrated that CREB expression correlates directly with the metastatic potential of murine melanoma cells (Rutberg et al., 1994) and that ATF-1 is not detected in normal melanocytes but is easily found in metastatic melanoma cells (Bohm et al., 1995). Whether these observations are causally related to tumorigenicity and metastasis of melanoma cells was not clear. To study the contribution of CREB and its associated proteins to tumor growth and metastasis of human melanoma cells, we have previously taken the approach of using a dominant-negative form of CREB, KCREB, that had been mutated in the DNA-binding domain (Walton et al., 1992). Expression of KCREB in melanoma cells decreased their tumorigenicity and metastatic potential in nude mice (Xie et al., 1997b). Moreover, in recent studies, we have demonstrated that CREB and its associated proteins act as survival factors for human melanoma cells, since quenching their activities by KCREB rendered the melanoma cells susceptible to apoptosis by agents that increase intracellular Ca2+ levels (Jean et al., 1998b). Collectively, these results indicate that CREB and its associated proteins (most likely ATF-1) play pivotal roles in the development of malignant melanoma.
Here, we focused our investigation on the role of ATF-1 on tumor growth and metastasis of human melanoma. To further investigate the cellular role of ATF-1 in melanoma progression, we hereby utilized current advances in the engineering of antibodies that have made possible the cloning of small single-chain Fv (ScFv) fragments. ScFv fragments contain the antigen-binding variable domains of the light and heavy chains connected by a peptide spacer (Raag and Whitlow, 1995; Winter and Milstein, 1991). When constructed in this manner, a single RNA transcript can be expressed and translated into an active protein that has the potential to interfere with the activity of targeted intracellular proteins. Intracellular ScFv fragments have been successfully employed to decrease the expression of ErbB-2 (Grim et al., 1996; Graus-Porta et al., 1995), the α subunit of human IL-2 receptor (Richardson et al., 1995), and to restore the transcription activity of mutant p53 (de Fromentel et al., 1999).
In studies aimed at addressing the role of ATF-1 in tumor growth and metastasis of human melanoma, a single chain Fv fragment (ScFv) was derived from a monoclonal antibody (mAb) anti-ATF-1. This mAb (designated mAb 41.4) has been shown to inhibit ATF-1 binding and transcriptional activation from CRE-containing promoters in vitro (Orten et al., 1994). In this study we provide evidence that expression of ScFv anti-ATF-1 in MeWo melanoma cells inhibited their tumorigenicity and metastatic potential in nude mice. Intracellular expression of ScFv anti-ATF-1 rendered the melanoma cells susceptible to apoptosis in vivo compared to control untransfected cells. These studies demonstrate for the first time that intracellular ScFv anti-ATF-1 can be used to quench ATF-1 activity not only as a method to explore its function but also as a modality for cancer therapy.
Expression of ScFv anti-ATF-1 in melanoma cells
MeWo melanoma cells were transfected with ScFv anti-ATF-1 expression vector. Expression of ScFv anti-ATF-1 in MeWo melanoma cells was analysed by RT–PCR utilizing specific primers corresponding to the light and heavy chains of the ScFv. Figure 1 shows that following transfection, we were able to isolate one pooled MeWo cells (designated MeWo-ScFv-P, lane 5) and 3 clones (designated as MeWo-ScFv-C4, C5, and C6, lanes 7, 8 and 9, respectively). No expression of ScFv anti ATF-1 was observed in control parental cells (lane 3) or in empty vector Neo-transfected cells (lanes 4 and 6). GAPDH was amplified simultaneously in the same tube to verify the integrity of the DNA and equal loadings. Clone MeWo-ScFv-C4 and the pooled cell line MeWo-ScFv-P were selected for further analyses to study the effect of ScFv anti-ATF-1 on tumor growth and metastasis of the transfected cells.
Effect of intracellular ScFv anti-ATF-1 on CRE-dependent promoter transactivation
To determine whether the ScFv anti-ATF-1 in the transfected cells was functional and capable of quenching the ATF-1 transcriptional activity, we analysed its ability to inhibit a CRE-dependent promoter. To that end, we used a CRE-dependent plasmid (Somat-BglII CAT), that contains the somatostatin gene promoter from −71 to +53 linked to the CAT reporter gene (Montminy et al., 1986), as we previously described (Jean et al., 1998b). We analysed the CAT activity driven by the Somat-BglII in MeWo-ScFv-P and in MeWo-ScFv-C4 in comparison to parental and control Neo-transfected cells (Figure 2). In the following transfection experiments, cell extracts were prepared and equivalent amounts of extracts exhibiting the same Renilla luciferase activity were tested for CAT activity. Figure 2 demonstrates that CAT activity driven by Somat-BglII promoter was inhibited by 10-fold in MeWo-ScFv-P (lane 5) as compared with control pooled Neo-transfected cells (lane 3). CAT activity in ScFv-C4 was inhibited by 35-fold (lane 6) as compared with the Neo-transfected clone (lane 4). The inhibition of CAT activity in ScFv-C4 was comparable to the inhibition observed in cells transfected with the dominant-negative construct of CREB, KCREB (35-fold, lane 7).
To make sure that the inhibition of ScFv anti-ATF-1 was specific for CRE-dependent promoters, we chose to work with a luciferase reporter gene expression vector driven by three AP-2 consensus response elements from the human metallothionein gene IIA ligated in front of a minimal TK promoter (3xAP-2Luc), that we described previously (Huang et al., 1998). There are no CRE's or CRE-like elements present, allowing for demonstration of specificity of the ScFv anti-ATF-1. Luciferase activity was not inhibited in the ScFv anti-ATF-1 melanoma-transfected cells as compared with parental and control Neo-transfected cells (Figure 3). Collectively these data indicate that expression of ScFv anti-ATF-1 in MeWo melanoma cells significantly and specifically inhibited the CRE-driven promoter.
Effect of intracellular ScFv anti-ATF-1 on tumor growth and metastasis of human melanoma cells
To determine the tumorigenicity of the ScFv anti-ATF-1 transfected cells, we injected 1×106 cells s.c. into BALB/c nude mice and tumor growth was monitored once a week for a period of 55 days. Both MeWo parental and Neo-transfected control cells (MeWo-Neo-C) grew progressively in all mice injected and reached 1.1–1.3 cm in mean diameter within 55 days (Figure 4). In contrast, MeWo cells expressing intracellular ScFv anti-ATF-1, both the pooled cell line (MeWo-ScFv-P) and clone MeWo-ScFv-C4 produced smaller tumors (0.45–0.5 cm in mean diameter). Growth of ScFv anti-ATF-1 transfected cells differed significantly from parental and Neo-control cells at 40, 45, 50 and 55 days after injections (P<0.01). These data indicate that intracellular ScFv anti-ATF-1 in MeWo cells suppressed their tumor growth in vivo.
In the next set of experiments, the metastatic potential of ScFv-transfected MeWo cells was determined in an experimental lung metastasis assay (Radinsky et al., 1994; Luca et al., 1997). To that end, BALB/c nude mice were injected i.v. with 1×106 MeWo-ScFv-transfected, parental, or Neo-control cells and 60 days later the number of lung metastases was counted. As shown in Table 1, parental and Neo-control cells produced lung tumor colonies in all injected mice with a median of >200. In contrast, cells expressing intracellular ScFv anti-ATF-1 produced fewer lung metastases with a median of 73 and 35 for MeWo-ScFv-P and MeWo-ScFv-C4, respectively.
To determine whether the inhibition of tumor growth and the decreased capacity to produce lung metastases by ScFv anti-ATF-1 in vivo was due to different growth rates in vitro, we compared their doubling time with control cells in culture. No difference in doubling time was observed in culture between the transfected and control cells (data not shown).
Intracellular ScFv anti-ATF-1 renders melanoma cells susceptible to apoptosis induction by thapsigargin
Since ATF-1 mediates both cAMP and Ca2+ transcriptional responses (Gonzales and Montiminy, 1989; Sheng et al., 1991) and since the induction of apoptosis by diverse exogenous signals is dependent on elevation of intracellular Ca2+ (McConkey and Orrenius, 1996), we decided to analyse if ATF-1 (and its associated proteins) could be involved in the resistance of melanoma cells to apoptosis induction, hence contributing to tumor growth and metastasis. To study the involvement of ATF-1 and its associated proteins in apoptosis, we used thapsigargin (Tg), which inhibits endoplasmic reticulum-dependent Ca2+-ATPase and thereby increases cytosolic Ca2+ (Thastrup et al., 1990). To that end, we next evaluated the ability of Tg to induce apoptosis in MeWo parental cells (MeWo-P), Neo-transfected cells (MeWo-Neo-P, MeWo-Neo-C), and ScFv anti-ATF-1-transfected clone (MeWo-ScFv-C4). As a control, we also analysed a KCREB-transfected clone (MeWo-KCREB-C8).
Apoptosis was determined by propidium iodide staining and FACS analysis. Figure 5A shows the results of a representative experiment after 48 h treatment with 1 μM Tg. The cells treated with Tg showed a hypodiploid DNA content indicative of apoptosis. The percentages of hypodiploid cells were higher in the cells expressing intracellular ScFv anti-ATF-1 (74.5 vs 17.9, 18.3 and 19.1% in parental and Neo-transfected cells). Apoptosis was also observed in the KCREB-transfected clone, MeWo-KCREB-C8, as we previously described (Jean et al., 1998b). Agents that stimulate adenylate cyclase or cAMP analogs (such as forskolin and dibutyryl-cAMP) did not induce apoptosis in the melanoma cells in our assay (data not shown), indicating that Tg-induced apoptosis was mediated via the Ca2+ and not the cAMP pathway. Figure 5B is a summary of at least three independent FACS analyses of propidium iodide staining.
We have previously demonstrated that treatment of melanoma cells with thapsigargin (Tg) caused phosphorylation of CREB and ATF-1 and activated their CRE-dependent transcription (Jean et al., 1998b). To verify that ScFv anti-ATF-1 expression inhibited the transactivation of a CRE-dependent promoter induced by Tg, we next analysed the CAT activity in the transfected cells after treatment with 1 μM Tg for 24 h. Figure 6 shows that CAT activity was increased by 15–20-fold in parental and Neo-transfected cells following treatment with Tg (lanes 2–4) when compared to untreated cells (lane 7). However, CAT activity driven by the Somat-BglII promoter was induced only by sixfold in MeWo-ScFv-P (lane 5) and by threefold in MeWo-ScFv-C4 (lane 6) in comparison to untreated cells (lane 7). These results show that ScFv anti-ATF-1 was able to suppress the fold induction of CRE-CAT activity induced by Tg.
Intracellular ScFv anti-ATF-1 induces apoptosis in vivo
We next determined whether the inhibition of tumor growth and metastasis by melanoma cells expressing ScFv anti-ATF-1 in nude mice was associated with apoptosis in vivo. We utilized the TUNEL method (Gavrieli et al., 1992) for identification of DNA fragmentation in cells undergoing programmed cell death in vivo, as we previously described (Radinsky et al., 1994). To that end, s.c. tumors were first established by injection of MeWo-P, MeWo-Neo-C and ScFv-C4 and 30 days later the TUNEL assay was performed on slides representing the resulting tumors. In each case the same area was selected. Figure 7 shows that no apoptosis was observed in MeWo parental cells, very few cells undergoing apoptosis were observed in the control neo-transfected cells, while a massive apoptosis was observed in the tumor cells expressing intracellular ScFv anti-ATF-1. Overall more areas of tumor cells undergoing apoptosis were observed in tumors produced by ScFv-transfected cells than in tumors produced by control cells (see ×4 magnification, in Figure 7B). We estimated that 70–80% of the tumor cells produced by ScFv-C4 cells underwent apoptosis in vivo as compared to 2–5% in tumors produced by the control cells. Here again, the tumors produced by ScFv-C4 cells were significantly smaller than the tumors produced by the control cells. These data suggest that the inhibition of tumor growth and eventually metastasis by human melanoma cells expressing ScFv anti-ATF-1 in nude mice is associated with apoptosis in vivo.
A possible mechanism for intracellular ScFv anti-ATF-1 activity
ScFv anti-ATF-1 is expressed in the cytoplasm and yet it has a profound effect on the activity of a nuclear transcription factor. One plausible explanation for the ScFv anti-ATF-1's action is that it binds to ATF in the cytoplasm, thus depleting them from the nucleus. To test this hypothesis, we used an immunostaining and confocal microscopy to localize the ATF-1 transcription factor in MeWo cells before and after transfection with ScFv anti-ATF-1. Figure 8 demonstrates that in control Neo-transfected cells (Figure 8A) ATF-1 was confined mainly to the nucleus (indicated by yellow), while in the ScFv-transfected cells, ATF-1 was observed with accumulation in the cytoplasm (indicated by green), and significant reduction of its levels in the nucleus (indicated by less yellow in the nucleus, Figure 8C). The field shown in the figure was chosen at random and is a representative of all fields examined. All ScFv anti-ATF-1 transfected cells exhibited an increase in ATF-1 expression in the cytoplasm and significant reduction in its levels in the nucleus as compared with control cells. Reduction of ATF-1 in the nucleus of ScFv-transfected cells was also confirmed by Western blot analysis (data not shown).
In this paper we demonstrate for the first time that inactivation of ATF-1 and its associated proteins in melanoma cells by means of ScFv anti-ATF-1 resulted in inhibition of their tumor growth and metastasis in vivo.
The expression of the transcription factors ATF-1 and CREB changes during the progression of human melanoma. CREB expression correlates directly with the metastatic potential of melanoma cells (Rutberg et al., 1994), and ATF-1 is not expressed in normal melanocytes but is easily found in metastatic melanoma cells (Bohm et al., 1995). As CREB and ATF-1 are implicated in cAMP and Ca2+-induced transcriptional activation, this upregulation in CREB and ATF-1 gene expression may confer growth advantage to melanoma cells in vivo by preventing them from undergoing apoptosis mediated through the Ca2+ pathway. Indeed, we have recently demonstrated that CREB/ATF-1 may act as survival factors for human melanoma cells (Jean et al., 1998b).
Here we demonstrate that intracellular expression of ScFv anti-ATF-1 in melanoma cells inhibits CRE-dependent promoter activation. This could be achieved by quenching the transcriptional activity of several members of the CREB transcription factors family including CREB, ATF-1 and ATF-2 (Ivanov and Ronai, 1999). Moreover, intracellular expression of ScFv anti-ATF-1 in melanoma cells resulted in inhibition of tumor growth and metastasis in nude mice. As ATF-1 and CREB act as survival factors for human melanoma cells (Jean et al., 1998b), here we show that intracellular expression of ScFv anti-ATF-1 in human melanoma cells rendered them susceptible to apoptosis in vivo, thus providing a mechanism for ScFv's action. Interestingly enough, under in vitro conditions apoptosis could be achieved following the treatment with thapsigargin that increases intracellular Ca2+. Under in vivo conditions, however, apoptosis was observed naturally, indicating the existence of apoptosis-inducing agents via the Ca2+ pathway within the immediate environment of the tumors. While the majority of the tumors produced by ScFv anti-ATF-1 expressing cells underwent apoptosis in vivo, some of the cells remained intact and managed to escape death by apoptosis, and continue to form tumors, albeit smaller in size. Two explanations may account for this phenomenon: (i) the cells that survived apoptosis may have lost the transfected gene in vivo or; (ii) the amount of apoptosis-inducing agents produced and secreted within the immediate environment of the tumor is not sufficient to produce 100% killing, especially within the core of the tumor. This was evident by small areas within the tumor that stained negative in the TUNEL assay.
The involvement of ATF-1/CREB in apoptosis has been documented in other cell systems. For example, Barton et al. (1996) demonstrated that T cell development is normal in transgenic mice that express KCREB. In contrast, thymocytes and T cells from those animals displayed a profound proliferation defect with subsequent apoptotic death in response to a number of different activation signals including Tg (Barton et al., 1996). In other studies, human monocytes infected with the influenza A virus died by apoptosis, which was associated with suppressed expression of CREB (Bussfield et al., 1997). In addition, using the same construct as in our studies, Hinrichs and associates have recently demonstrated that intracellular expression of ScFv anti-ATF-1 in clear cell sarcoma caused apoptosis in these cells in tissue culture (Bosilevac et al., 1999). Taken together, these studies and our present results suggest that ATF-1/CREB act as negative regulators of apoptosis in diverse types of cells. While we consider apoptosis as the main mechanism by which ScFv anti-ATF-1 inhibits tumor growth and metastasis of human melanoma cells, we cannot rule out the possibility that quenching of ATF-1/CREB may influence the expression of other genes involved in the progression of human melanoma which harbor CRE element in their promoter.
One mechanism by which ATF-1/CREB proteins might rescue cells from apoptosis is by upregulation of Bcl-2 expression. Indeed, ATF-1/CREB have been shown to function as positive regulators of the Bcl-2 gene via a direct binding to the CRE element within the Bcl-2 promoter (Wilson et al., 1996). The CRE site in the Bcl-2 promoter appears to play a major role in the induction of Bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis (Wilson et al., 1996). In our studies, we did not observe any changes in the expression of Bcl-2 or Bcl-2 related proteins (Bcl-x, Bax and Bad) in control or ScFv-transfected cells following Tg treatment (data not shown). We also did not observe any changes in the expression of the known caspases nor in the expression of p53 (data not shown). Thus, the mechanism by which ATF-1/CREB act as survival factors and rescue melanoma cells from apoptosis induced by agents that increase intracellular Ca2+ remains to be elucidated. Exploring the possible downstream targets of CREB/ATF-1 signaling that are important for the increased survivability of melanoma cells is now under investigation in our laboratory.
Two plausible explanations for the effect of ScFv anti-ATF-1 have been considered; either ScFv anti-ATF-1 inhibits DNA binding of ATF-1 and CREB in the nucleus of cells, or alternatively, the inhibitory effect of ScFv may be due to the removal of ATF-1 from the nuclear pool by altering its intracellular processing or nuclear localization. Our results support the second explanation as the mechanism of its action. Based on immunostaining coupled with confocal microscopy and Western blot analyses, it seems that intracellular expression of ScFv anti-ATF-1 in melanoma cells caused a reduction of ATF-1's levels in the nucleus. These results indicate that ScFv anti-ATF-1 is capable of quenching ATF-1's transcriptional activity even in the absence of nuclear localization signal (NLS). However, we cannot rule out the possibility that some of the ScFv anti-ATF-1 will be transported into the nucleus with ATF-1/CREB serving as carriers, as was found to be the case for ScFv-p53 (de Fromentel et al., 1999).
Recently, ScFvs have been used to achieve phenotypic knockout of target proteins involved in neoplasia such as Ki-ras, ErbB-2, EGF-R and IL-2R (Graus-Porta et al., 1995; Marasco, 1995; Griffiths et al., 1993). ScFvs were also used to restore the transcriptional activity of mutant p53 (de Fromentel et al., 1999). These studies provide a proof of principle for targeted disruption of oncogenic proteins by ScFvs in an in vitro model. Our studies, on the other hand represent the first in vivo evidence that ScFv anti-ATF-1 can be used as a modality for cancer therapy.
The therapeutic modalities to control tumor growth and metastasis of human melanoma are very limited (Bar-Eli, 1997). The idea of using ScFv anti-ATF-1 to control tumor growth and metastasis of human melanoma is especially appealing since a specific expression of ScFv anti-ATF-1 in melanoma cells could be achieved by placing the ScFv-ATF-1 expression under the control of the tyrosinase promoter (Vile et al., 1994).
Materials and methods
The human melanoma MeWo cell line was established in culture from a lymph node metastasis of a melanoma patient (Fogh et al., 1997), and was kindly provided to us by Dr S Ferrone (New York Medical College, New York, NY, USA). MeWo cells are tumorigenic and metastatic in nude mice (Ishikawa et al., 1988). Cells were maintained in culture as adherent monolayers in MEM, supplemented with 10% fetal bovine serum (Summit, Ft Collins, CO, USA), sodium pyruvate, nonessential amino acids, L-glutamine and penicillin-streptomycin (Life Technologies Inc., Grand Island, NY, USA). The transfected cells MeWo-Neo-P, MeWo-Neo-C, MeWo-ScFv-P, MeWo-ScFv-C4 and MeWo-KCREB-C8 were maintained in the same medium contained G418 (Life Technologies Inc.) at 1 mg/ml. All cells were grown at 37°C with 5% CO2.
Animals and tumor cell injection
Male athymic nude mice (BALB/c background) were obtained from the Animal Production Area of the NCI-Frederick Cancer Research and Development Center (Frederick, MD, USA). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used at 8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the US Department of Agriculture, Department of Health and Human Services, and the National Institutes of Health. Their use in these experiments was approved by the institutional Animal Care and Use Committee.
To prepare tumor cells for inoculation, cells in exponential growth phase were harvested by brief exposure to 0.25% trypsin, 0.2% EDTA solution (w/v). The flask was sharply tapped to dislodge the cells, and supplemented medium was added. The cell suspension was pipetted to produce a single-cell suspension. The cells were washed and resuspended in Ca2+- and Mg2+-free HBSS to the desired cell concentration. Cell viability was determined by Trypan blue exclusion, and only single-cell suspensions of more than 90% viability were used. Subcutaneous tumors were produced by injecting 1×106 tumor cells in 0.2 ml of HBSS over the right scapular region as we previously described (Jean et al., 1998a; Luca et al., 1995). Growth of subcutaneous tumors was monitored for 55 days. For experimental lung metastasis, 1×106 tumor cells in 0.2 ml of HBSS were injected into the lateral tail vein of nude mice. The mice were killed after 60 days, and the lungs were removed, washed in water, and fixed with Bouin's solution for 24 h to facilitate counting of tumor nodules as we described previously (Radinsky et al., 1994; Singh et al., 1995; Luca et al., 1997). The number of surface tumor nodules was counted under a dissecting microscope. Sections of the lungs were stained with hematoxylin and eosin to confirm that the nodules were melanoma and to identify micrometastasis.
RNA preparation and RT–PCR
Total cellular RNA was prepared from 60 mm culture dishes of 70–80% confluent cells using TRIzol reagent (Life Technologies Inc.) according to manufacturer's instructions. 1 μg of total RNA was reverse-primed with an oligo poly-dT primer and extended with MMLV reverse transcriptase (Clontech, Palo Alto, CA, USA). Using Advantage cDNA PCR kit (Clontech), cDNA was used for PCR amplification with primers specific of the ScFv-ATF-1 fragment (5′-GGAGTGGGTCGCATACATTA-3′ and 5′-AAGATGAGGAGTTTGGGTGG-3′) and primers of GAPDH (5′-GAGCCACATCGCTCAGAC-3′) and 5′-CTTCTCATGGTTCACACCC-3′). The PCR reaction was carried out in a Perkin Elmer thermal cycler (Foster City, CA, USA). The reaction was submitted to an initial denaturation step (2 min at 96°C) followed by 27 cycles of denaturation (1 min 94°C), annealing (1 min 57°C), and extension (1 min 72°C) then followed by an extension time of 5 min 72°C. PCR products were analysed on a 3% agarose gel containing ethidium bromide.
The pCMV-ScFv-ATF-1 that contains the cDNA coding for the ScFv fragment specific for the transcription factor ATF-1 (ScFv-ATF-1) has been previously described (Bosilevac et al., 1998). ScFv-ATF-1 cDNA was first subcloned in pBluescript II S+ (Stratagene, San Diego, CA, USA) using BglII/BamHI and NotI. PRSV-ScFv-ATF-1 was generated by placing the cDNA of ScFv-ATF-1 into the multiple cloning site of pRc/RSV (Invitrogen, San Diego, CA, USA) using HindIII and NotI. pRSV-KCREB was kindly provided by Dr Richard H Goodman (Oregon Health Sciences, Portland, OR, USA). PRSV-KCREB contains a full length CREB cDNA with a single base pair substitution in the DNA-binding domain that causes a change from Arg287 to Leu (Walton et al., 1992). In control experiments, a pRc/RSV (Invitrogen) construct that lacks ScFv-ATF-1 or KCREB cDNA was used. The CRE-dependent plasmid (Somat-BglII) was obtained from Dr. Marc R. Montminy (Harvard Medical School, Boston, MA, USA). The Somat-BglII CAT construct contains the somatostatin gene promoter from −71 to +53 linked to the chloramphenicol acetyltransferase (CAT) reporter gene (Montminy et al., 1986). The pRL-β-actin vector contains the Renilla luciferase reporter gene (Promega) which is driven by the β-actin promoter. The β-actin promoter does not contain CRE element. The AP-2 binding site-luciferase reporter (3xAP-2Luc) construct contains an AP-2-dependent promoter (without CRE) linked to the Firefly luciferase reporter gene (Huang et al., 1998; Jean et al., 1998a).
Stable transfection of melanoma cells with ScFv-ATF-1
5×105 MeWo cells were transfected with 5 μg of pRSV-ScFv-ATF-1 expression vector, pRSV-KCREB expression vector or control pRc/RSV vector using 20 μl of Lipofectin reagent (Life Technologies Inc.). Transfections were carried out according to the manufacturer's instructions. At 5 h following transfection, the medium was changed to normal growth medium, and the cells were then further incubated for 48 h at 37°C. Cells were selected after 48 h with standard medium containing G418 at 1 mg/ml (Life Technologies, Inc.). Two-to-three weeks later, G418-resistant colonies were isolated by trypsinization using a penicylinder and established in culture to obtain clones. Pools of G418-resistant cells were also prepared as we previously described (Xie et al., 1997a,b).
Melanoma cells (2×105) were transfected with 3 μg of the basic CAT expression vector with no promoter/enhancer sequence (pCAT-Basic) from Promega or the Somat-BglII CAT construct using 20 μl of lipofectin according to the manufacturer instructions (Life Technologies, Inc.). Fifty nanograms of pRL-β-actin vector was introduced in each experiment to normalize for variations in transfection efficiency. After 5 h, the transfection medium was replaced by normal growth medium, and the cells were further incubated for 48 h at 37°C. For Thapsigargin treatment, Thapsigargin (1 μM) was added to the culture medium 24 h after transfection and the cells were incubated for an additional 24 h. The transfected cells were washed twice with PBS and lysed in the Reporter Lysis Buffer (Promega). After removal of cell debris by centrifugation, cell extracts were first assayed for Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). Samples were normalized as the same relative luciferase activity and assayed for CAT activity (Gorman et al., 1982) as we previously described (Hudson et al., 1995). Briefly, cell extracts were incubated with 0.025 mCi of [14C]chloramphenicol and 0.35 mM acetyl coenzyme A in 25 mM Tris-HCl pH 7.9 for 2 h at 37°C. The nonacetylated and acetylated chloramphenicol were extracted in ethyl acetate and separated by thin-layer chromatography with chloroform/methanol (95 : 5). The chromatograph was exposed to X-ray film. The conversion of chloramphenicol to the acetylated form was quantified by scanning densitometry of an autoradiograph with personal densitometer (Molecular Dynamics, Sunnyvale, CA, USA). Each assay was repeated at least three times; there was less than 10% variation among individual transfections.
Melanoma cells (2×105) were transfected with 3 μg of the 3xAP-2Luc and 50 ng of pRL-β-actin vector to normalize for transfection efficiency by using 20 μl of Lipofectin reagent (Life Technologies Inc.). At 5 h following transfection, the medium was changed to normal growth medium, and the cells were further incubated for 48 h at 37°C. The cells were then washed with PBS and harvested in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activities were normalized for Renilla luciferase activities. Results are reported as the average of at least three independent experiments.
Detection of apoptosis by propidium iodide-staining and flow cytometry
Cells were plated overnight in culture medium described above without G418 and incubated in the presence of 1 μM Thapsigargin (Tg) for 48 h. All cells (including those which had detached and those remaining adherent) were collected, and adherent cells were harvested in PBS containing 0.1% EDTA. Cells were washed twice with PBS, pelleted by centrifugation, and resuspended in PBS containing 3 mM Sodium Citrate, 0.1% Triton X-100 and 50 μg/ml propidium iodide (PI) and incubated 2–4 h. The propidium iodide-stained cells were subjected to flow cytometric analysis on an EPICS Profile flow cytometer (Coulter Corp., Miami, FL, USA). Results are reported as the average of at least three independent experiments as reported previously (Jean et al., 1998b).
Detection of apoptosis by TUNEL
Sections of subcutaneous tumors, characterized by the same size, were prepared and stained for apoptosis by in situ labeling of DNA breaks using terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method (Gavrieli et al., 1992). Following deparaffinization and hydration, tumor sections were incubated with proteinase K (20 μg/ml) for 15 min at room temperature (RT), and washed. Endogenous peroxydase was inactivated with 2% H2O2 for 5 min at RT. Tumor sections were rinsed with water and immersed in TdT buffer (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). Tumor sections were then covered with TdT (0.3 units/ml) and dUTP-biotin in TdT buffer and incubated in a humidified chamber at 37°C for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 μM sodium chloride, 30 mM sodium citrate) for 15 min at RT. Tumor sections were rinsed with water, covered with a 2% BSA solution for 10 min, rinsed in water, immersed in PBS for 5 min, covered with Strep-Avidin Peroxydase (Dako Corp., Carpinteria, CA, USA), diluted 1 : 400 in buffer, and incubated for 30 min at 37°C. The sections were washed, immersed in PBS for 5 min and stained with 3-amino, 9-ethylcarbazole (AEC) for 30 min at 37°C. For positive control, each tumor section was treated with DNase at 1 ng/ml (Life Technologies Inc.) for 10 min in buffer (30 mM Trizm base, pH 7.2, 104 mM sodium cacodylate, 4 mM MgCl2, 0.1 mM DTT). For negative control, TdT and dUTP-biotin were omitted in the procedure.
Immunofluorescence staining and confocal microscopy
Cells were plated (1×105) in each chamber of a two-chamber Falcon glass slide (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Cells were grown to 60% confluence in MEM+10% FBS and prepared for immunofluorescent staining. Briefly, the medium was decanted and cells were fixed with 4% paraformaldehyde for 20 min at 4°C and washed with PBS two times for 5 min. Cells were permeabilized with 0.2% Triton X-100 for 10 min at 4°C and washed with PBS for 5 min. Cells were incubated with blocking buffer (10% goat serum and 5% normal horse serum in PBS) for 10 min followed by incubation with A100 anti-ATF-1 in blocking buffer (1 : 100) overnight at 4°C. Antibody to ATF-1 (A100) was a gift from Dr Michael R Green (University of Massachusetts Medical Center, Worcester, MA, USA) (Liu et al., 1993). Slides were washed with PBS two times for 5 min and incubated with FITC-labeled secondary antibody (anti-rabbit Ig) in blocking buffer (1 : 200) for 2 h at room temperature. Imaging was done using a Zeiss confocal laser scanning microscope (upright version) using an argon laser (488 nm, power 10 mW). Signals were collected by photomultipliers with 590 nm (PI) long pass filter and 520–560 nm (FITC) band pass filter, respectively. Digitized images were transmitted to a Macintosh-based image analysis system through a GPIB interface using BDS-LSM software (Biological Detection Systems, Pittsburgh, PA, USA). Composite images were assembled using Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA, USA).
The significance of the in vitro results was determined by the Student's t-test (two-tailed); the significance of the in vivo metastasis results were determined by the Mann–Whitney U test.
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We thank Dr MR Montiminy for the gift of the CRE-dependent promoter-CAT vector, Dr MR Green for the antibody against ATF-1, Dr RH Goodman for the KCREB expression vector, and Patherine Greenwood for expert preparation of this manuscript. Supported in part by NIH grant CA76098 (to M Bar-Eli).
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Jean, D., Tellez, C., Huang, S. et al. Inhibition of tumor growth and metastasis of human melanoma by intracellular anti-ATF-1 single chain Fv fragment. Oncogene 19, 2721–2730 (2000). https://doi.org/10.1038/sj.onc.1203569
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