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Tumor Biology |
Section of Thoracic Molecular Oncology, Departments of Thoracic and Cardiovascular Surgery [L. J., M. N., K. X., N. Y., B. F., J. A. R.] and Biomathematics [E. N. A.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Internal Medicine and Pharmacology, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390 [B. G., D. B., M. K., C. K., J. D. M.]; and Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [M. I. L.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of nested 3p21.3 homozygous deletions in small cell lung cancers and a breast cancer line directed positional cloning efforts to a 630-kb region, which was narrowed subsequently to a 120-kb subregion by a breast cancer homozygous deletion (10
, 11)
. This defined 3p21.3 region undergoes allele loss in 
80% of primary lung cancers and 40% of preneoplastic or normal epithelial samples of smoking-damaged lung, marking it as one of the first sites to be involved (6)
. In addition, patients whose peripheral blood lymphocytes showed greater damage in this 3p21.3 region after in vitro treatment with the carcinogen benzo-a-pyrene diol epoxide had an increased risk of having lung cancer, suggesting the potential for genetic polymorphisms in this region predisposing to lung cancer development (12)
. The 630-kb region contains 
25 genes, whereas 9 genes are located in or on the border of the 120-kb 3p21.3 region (10)
. This group of potential TSGs includes CACNA2D2 (GenBank no. AF040709), PL6 (U09584), 101F6 (AF040704), NPRL2 (AF040707), FUS1 (AF055479), BLU (U70880), RASSF1 (AF040703, RASSF1C and AF102770, RASSF1A), HYAL2 (U09577), and HYAL1 (U03056). The RASSF1A isoform of the RASSF1 gene has been studied extensively for promoter methylation in a variety of tumors, including lung and breast cancer, found to be frequently epigenetically inactivated in these tumors, and shows the ability to suppress lung cancer malignant growth (13
, 14)
. The FUS1 gene was also found to suppress the growth of NSCLCs in vitro (15)
. However, there have been no detailed tests comparing the activity of several of the genes in this small region or in testing their effect on lung cancer xenografts (local tumors or metastases) in vivo. At the start of the search for a 3p21.3 TSG, everyone expected that one gene would be found that frequently suffered mutations. However, from detailed studies of the genes in the region, that was not the case (10)
. In addition, the possibility of haploinsufficiency needed to be considered. Thus, it was important to further define the tumor suppressing capability of these genes both in vitro and in vivo. Such identification, which is the focus of the current report, would target the gene(s) for development as new tools for the early detection, monitoring of prevention efforts, prognosis, and therapy of lung and other cancers.
In this study, we used recombinant adenoviruses to introduce WT 3p21.3 genes into NSCLC tumor cell lines or tumor xenografts, where 3p21.3 120-kb region genes are either heterozygous or homozygous to characterize their potential tumor suppressing function in vitro and in vivo. We demonstrate that introduction of individual WT 3p21.3 genes by recombinant adenoviral vector-mediated transfer into lung cancer cells with loss of heterozygosity at the3p21.3 120-kb region inhibited tumor cell growth and induced apoptosis in vitro. Moreover, intratumoral injection of recombinant adenoviral vectors containing WT 3p21.3 genes significantly suppressed growth of human NSCLC xenografts, whereas systemic administration of protamine-complexed adenoviral vectors of 3p21.3 genes efficiently inhibited development of experimental metastases of lung cancer cells in xenograft mouse models. Together, our results strongly suggest that multiple contiguous genes in the 3p21.3 chromosomal region may function as TSGs in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of Recombinant Ad-3p21.3 Gene Vectors.
The recombinant Ad-3ps were constructed using our recently developed ligation-mediated plasmid adenovirus vector construction system, named herein pAd-RAP and pAd-RAP-Shuttle (detailed structures of plasmids will be provided on request). The 3p21.3 genes were assembled as a mammalian expression cassette that is driven by a cytomegalovirus promoter and tailed with Bovine Growth Hormone poly(A) signal sequence. The resulting Ad-3p vectors were named Ad-101F6, Ad-NPRL2, Ad-BLU, Ad-RASSF1C, Ad-FUS1, Ad-HYAL1, and Ad-HYAL2. Sequences of 3p21.3 genes in the viral vectors were confirmed by automated DNA sequencing. A vector expressing GFP gene (Ad-GFP), and a vector carrying the ß-galactosidase gene LacZ (Ad-LacZ), were used to monitor efficiency of transduction by the viral vectors and as nonspecific transgene expression controls. Ad-EV, an E1-deleted empty vector, was used as a negative control. Ad-p53, a vector containing the WT p53 gene, was used as a positive tumor suppressor control. Viral titers were determined by both absorbance measurement (i.e., vp/ml) and plaque assay (i.e., pfu/ml).
Cell Viability Assay.
Inhibition of tumor cell growth by treatment with various Ad-3p vectors was analyzed by quantitatively determining cell viability using an improved 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Molecular Biochemicals, Indianapolis, IN; Ref. 17
) as described previously (18)
. Percentage of cell viability was calculated in terms of the absorbency of treated cells relative to the absorbency of untreated control cells. Experiments were repeated at least three times with quadruplicate samples for each treatment in each individual experiment.
Analysis of Apoptosis and Cell Cycle Kinetics.
Induction of apoptosis in tumor cells treated by various Ad-3p vectors was analyzed by flow cytometry (FACS) using TUNEL reaction with FITC-labeled dUTP (Roche Molecular Biochemicals, Mannheim, Germany). Cells were processed for FACS analysis for apoptosis and cell cycle kinetics as described previously (19)
.
Animal Studies.
All animals were maintained, and animal experiments were performed under NIH and institutional guidelines established for the Animal Core Facility at the University of Texas M. D. Anderson Cancer Center. Procedures for A549 and H1299 s.c. tumor inoculations in nu/nu mice were described previously (19)
. When average tumor size reaches 0.5 cm in diameter, mice were injected intratumorally three times within a week with various Ad-3p and control vectors at a dose of 3 x 1010 pfu (3 x 1012 vp)/tumor in a volume of 0.2 ml. Differences in tumor volumes between treatment groups were analyzed with a mixed model ANOVA using the Statistica software (StatSoft, Inc., Tulsa, OK). A difference was considered to be statistically significant when P < 0.05.
An experimental A549 lung metastasis model was used to study the effects of 3p21.3 genes on development of metastases. Briefly, nu/nu mice were inoculated with A549 cells (1–2 x 106) in 200 µl of PBS via tail vein injection. Pulmonary experimental metastatic tumor colonies were formed 7–10 days after inoculation. Then, protamine-complexed Ad-3p (P-Ad3p) vectors or control complexes were administered systemically to animals by i.v. injection for three times within a week at each a dose of 2–5 x 1010 vp/200–500 µg of protamine in a total volume of 200 µl/animal. The P-Ad complexes are prepared by mixing an equal volume of the adenoviral vector (1 x 1010 vp) and the protamine sulfate (100 µg; Fujisawa USA, Inc., Deerfield, IL) in room temperature for 15 min and then bringing it to a total volume of 200 µl with PBS. Two weeks after the last injection, the animals were euthanized, and their lung metastatic tumors were stained with India ink. Tumor colonies on lung surfaces were counted under a dissecting microscope without knowledge of the treatment groups, and the lung tissues were sectioned for further pathologic and immunohistochemical analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100:1. The transduction efficiency was determined by examining GFP-expressing cells in the Ad-GFP-transduced cell population under a fluorescence microscope and was found to be >80% at the highest MOI applied for each cell line.
Cell proliferation was analyzed by using the XTT assay to determine the number of viable cells at 1, 2, 3, and 5 days after transduction (only data for day 5 at various MOIs are shown; Fig. 1
). In all cases, the viability of transduced cells was compared with that of untransduced (PBS treated) control cells (whose viability was set at 100%). As can be seen in Fig. 1
, cell viability was reduced significantly in Ad-101F6-, Ad-Fus1-, and Ad-NPRL2-transduced A549 and H460 cells, which show homozygosity for multiple 3p21.3 markers and contain WT p53, and H1299 cells, which exhibit 3p21.3 homozygosity but also have a homozygous deletion of p53. A modest reduction of cell viability was shown in Ad-RASSF1C-transduced H1299 cells (data not shown). However, no significant effect on growth was observed in any of these cells transduced with Ad-HYAL1, Ad-HYAL2, Ad-BLU, Ad-EV, or Ad-LacZ. These results suggest that exogenous expression of some but not all WT 3p21.3 genes can inhibit 3p-deficient tumor cell growth in vitro.
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. As shown in Fig. 1
, HBECs transduced with all Ad-3p genes at MOIs that generated >80% transduction efficiency had reductions in cell number after 5 days of transduction of <10%, whereas H358 cells transduced with the same vectors had losses of <20% when compared with the untransduced control cells. Similar levels of losses of cell numbers were observed in H358 and HBEC cells transduced with control vectors Ad-EV and Ad-LacZ. As a positive control, H358 cells, which are homozygously deleted for p53, showed reduced cell numbers when transduced with the Ad-p53 vector. These results, coupled with the lack of effect with Ad-LacZ, Ad-HYAL2, Ad-HYAL1, Ad-RASSF1C, and Ad-BLU, demonstrate the specificity of the tumor suppressing function of 3p21.3 genes FUS1, NPRL2, and 101F6 in 3p-deficient tumor cells and indicate that no generalized cytotoxicity was associated with exogenous expression of these WT 3p21.3 120-kb region genes.
Expression of 3p21.3 genes in Ad-3p-transduced H1299 and normal HBEC cells was verified by quantitative real-time reverse transcription-PCR. Known concentrations of human Raji total RNA and a TaqMan probe for glyceraldehyde-3-phosphate dehydrogenase cDNA were used to make a standard curve as an internal control. The increase in levels of transcripts of exogenously expressed 3p21.3 genes relative to those of endogenously expressed mRNAs in HBEC is 10–15-, 30–50-, and 50–80-fold at an MOI of 100, 500, and 1000 vp/cell, respectively (data not shown). The levels of expression in transduced H1299 cells were similar to those in HBEC (data not shown). In addition, there was an association between increased levels of expression of these 3p21.3 genes and increased MOIs of the corresponding Ad-3p vectors transduced in both H1299 and HBEC cells. The expression of FUS1 and 101F6 proteins after transfection was detected by Western blot analysis using available polyclonal antibodies raised against the oligopeptides derived from their deduced amino acid sequences (data not shown).
Induction of Apoptosis by 3p Genes in Ad-3p-transduced Tumor Cells.
The ability of exogenously expressed 3p21.3 genes to induce apoptosis in the Ad-3p-transduced H1299, A549, H460, H358, and HBEC cells was analyzed by FACS using the TUNEL reaction (Fig. 2)
. Induction of apoptosis was detected in Ad-101F6-, Ad-FUS1-, and Ad-NPRL2-transduced A549 (Fig. 2A)
, H1299 (Fig. 2B)
, and H460 (Fig. 2C)
cells but not in H358 (Fig. 2D)
and HBEC (Fig. 2E)
cells. The apoptotic cell populations increased with increased duration of transduction; >15–20, 40–65, and 75% of cells were apoptotic 5 days after transduction with Ad-101F6, Ad-FUS1, and Ad-NPRL2 in the transduced H1299, A549, and H460 cells, respectively, whereas only 7 and 10% of cells treated with PBS alone and transduced with Ad-EV vector, respectively, were TUNEL positive after the same time interval. The levels of apoptosis induction by Ad-101F6, Ad-FUS1, and Ad-NPRL2 were quantitatively greater in A549 and H460 cell lines with WT p53 genes (Fig. 2, A and C)
than that in H1299 cell line deleted for p53 gene (Fig. 2B)
. Levels of apoptosis in A549 and H460 cells were comparable with those induced by Ad-p53 in p53-deficient H1299 and H358 cells (Fig. 2, B and D)
. However, no significant induction of apoptosis was observed in any tumor cell line transduced by Ad-BLU, Ad-RASSF1, Ad-HYAL2, and Ad-HYAL1 (Fig. 2)
. The levels and time of induction of apoptosis in cells transduced by these Ad-3p vectors correlated with those of reduction of cell numbers in cells treated with the same vectors (Fig. 1)
, suggesting that suppression of tumor cell growth by these 3p21.3 genes is mediated directly or indirectly through a mechanism of apoptosis.
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. The growth of tumors was recorded from the first injection until 23 days after the last injection. Tumor volumes were normalized by calculating the percentage increase in tumor volume after treatment relative to volume at the beginning of treatment in each group. In both A549 (Fig. 3A)
and H1299 (Fig. 3B)
tumor models, all of the tumors treated with Ad-101F6, Ad-FUS1, or Ad-NPRL2 showed significantly suppressed growth (P < 0.001), compared with mouse groups treated with Ad-LacZ or Ad-EV controls, whereas no significant effect was observed in Ad-HYAL1-, Ad-BLU (data not shown)-, and Ad-HYAL2 (data not shown)-treated tumors. Moreover, a significantly stronger inhibition of tumor growth was shown in A549 tumors treated with Ad-101F6 and Ad-NPRL2 vectors than in tumors treated with the Ad-p53 vector (Fig. 3A)
.
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. The adenoviral 3p21.3 gene vectors were complexed to protamine and delivered via i.v. injection. The development of A549 pulmonary metastases was inhibited significantly, and the numbers of metastatic tumor colonies found on the surfaces of lungs from mice inoculated with A549 cells were reduced by >80% in animals treated with P-Ad-101F6, P-Ad-FUS1, P-Ad-NPRL2, or P-Ad-HYAL2 (70% reduction), compared with those in control treatment groups (Fig. 4)
. However, no significant reduction of metastatic colony formation was observed in animals treated with P-Ad-HYAL1 and P-Ad-BLU. These data are consistent with results obtained from Ad-3p-treated s.c. tumor xenografts, further supporting the roles of these 3p21.3 genes in suppression of tumor growth and inhibition of tumor progression in vivo. Finally, we noted no systemic toxicity to the mice given the systemic injection of PAd-3p complexes.
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DISCUSSION |
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-irradiation of human glioblastoma cells (21
, 22)
. The level of 3p21.3 gene expression by the adenoviral vector-mediated 3p21.3 gene transfer in normal HBEC cells was increased 10–15-fold (data not shown) and is close to the elevated levels of TSG expression induced by environmental stimuli under physiological conditions shown by that of p53. In most cases, there is loss of heterozygosity at the 3p21.2 locus with no mutations detected in the remaining allele. However, haploinsufficiency can be associated with abrogation of tumor suppressor activity, e.g., the p27 gene is haploinsufficient for tumor suppression with tumor suppressor activity critically dependent on the absolute level of p27 protein expression (23) . Elevated p27 expression inhibits cell cycle progression and promotes apoptosis in human glioma, colon, NSCLC, and mantle cell lymphoma, suggesting that p27 acts as a rheostat rather than as an on/off switch tumor suppressor in suppressing neoplasia (24) . Similar to p27, some of the 3p21.3 genes are possibly inactivated by haploinsufficincy (10) , and the modulation of protein expression may play an important role for their tumor suppressor activities. Furthermore, the overexpression of these 3p21.3 genes at higher MOIs may be pharmaceutically appropriate for enhancing their function as cancer therapeutics and may be necessary for proper TSG function to overcome degradation pathways and inactive pathways in the cancer cell. The selectivity of the vectors with respect to growth inhibition and induction of the specific pathway of apoptosis in cancer compared with normal cells further supports their physiological role.
Inhibition of cell proliferation and induction of cell death by activated TSGs, such as retinoblastoma (Rb) and p53, are attributed primarily to these genes’ ability to mediate cell cycle arrest and apoptosis (25, 26, 27) . Because apoptosis is a genetically programmed cellular response to environmental stresses or stimuli, inactivation of TSGs involved in the apoptotic pathways could result in deregulated cell proliferation and tumorigenesis. To elucidate the mechanism governing the inhibition of NSCLC cell growth by 3p21.3 genes, we studied the effects of exogenously expressed 3p21.3 genes on apoptosis mediated by adenoviral vector transduction. Introduction of WT 3p21.3 genes 101F6, FUS1, and NPRL2 into the 3p-deficeint A549, H1299, and H460 cells induced apoptosis. However, this was not a generalized feature of 3p21.3 gene overexpression, as the HYAL2, HYAL1, and BLU genes from this same 120-kb region did not induce a significant increase in apoptosis in the same lung cancer cells. The time and the magnitude of the induction of apoptosis by these 3p21.3 genes were also well correlated with those of the inhibition of growth observed in vitro. These observations suggest that the tumor suppressing function mediated by the 3p21.3 genes is through induction of apoptosis.
To demonstrate whether the observed inhibitory effects of these 3p21.3 genes on tumor cell growth in vitro could be reproduced in vivo, we evaluated the efficacy of 3p21.3 genes in suppressing tumor growth by directly injecting Ad-3p vectors into A549 or H1299 tumor xenografts in nu/nu mice. Growth of both A549 and H1299 tumors was suppressed significantly by treatments with Ad-101F6, Ad-FUS1, and Ad-NPRL2. Furthermore, we explored the tumor suppressing potential of 3p21.3 genes in inhibiting experimental metastases in vivo by systemic administration of protamine-Ad-3p complexes. The novel protamine-Ad-3p complexes developed as part of this study allowed us to deliver 3p21.3 genes efficiently to the lung by systemic injection. The development of metastases was inhibited effectively by the protamine-adenovirus complex-mediated transfer of the 101F6, FUS1, HYAL2, and NPRL2 genes. These in vivo data are consistent with the in vitro data, further supporting the roles of 3p21.3 genes as TSGs.
Two of the 3p21.3 genes, HYAL1 and RASSF1C, showed neither tumor suppressor activity in vitro nor in vivo nor apoptosis-inducing activity in vitro in all cell lines tested. Recently, one splicing isoform of one of the genes, RASSF1A, has been shown to undergo epigenetic inactivation by promoter region hypermethylation acquired in tumors. This isoform, but not the expressed RASSF1C isoform, also exhibits functional tumor suppressing activity. Consistent with these results, we also found no significant effects on growth of NSCLC cells and induction of apoptosis in these NSCLC cells in vitro and in vivo in our experiments using the Ad-RASSF1C vector (data not shown). The results with RASSF1A indicate that it will be important to study all of the genes in the region for loss of expression via tumor-acquired promoter hypermethylation. Alternatively, with 3p allele loss, haploinsufficiency of one or more of these 3p21.3 genes may play a role in tumorigenesis. On the basis of the evidence that multiple contiguous 3p21.3 genes, including 101F6, NPRL2, RASSF1A, and FUS1, exhibited varied degrees of tumor suppressing activity, we propose that genes in this 3p21.3 120-kb region act together as part of a tumor suppressor region to suppress tumor growth through their functional activation of tumor suppressing pathways. Likewise, their inactivation may contribute directly to the development of cancer because of haploinsufficiency, loss of expression, or rarely mutations. In summary, we have demonstrated here for the first time that introduction of several WT 3p21.3 genes (101F6, NPRL2,and FUS1) contiguously located within a 120-kb region by recombinant adenoviral vector-mediated gene transfer into 3p-deficient tumor xenografts and tumor cell lines efficiently suppressed tumor cell growth and metastases and induced apoptosis in vitro and in vivo. These results suggest the role of these genes as TSGs. A better understanding of the biological function of genes in this region may result in the development of new strategies for the prevention, early detection, diagnosis, and treatment for lung cancer and other human cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the National Cancer Institute and the NIH SPORE (2P50-CA70970-04, to J. D. M. and J. A. R.; P01 CA78778–01A1, to J. A. R.; and CA71618, to J. D. M.), gifts to the Division of Surgery from Tenneco and Exxon for the Core Laboratory Facility, the UT M. D. Anderson Cancer Center Support Core Grant (CA 16672), a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature (Project 8), a W. M. Keck Gene Therapy Career Development Grant (L. J.), and a sponsored research agreement with Introgen Therapeutics, Inc. (SR93-004-1). 
2 To whom requests for reprints should be addressed, at Department of Thoracic and Cardiovascular Surgery, Box 445, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 745-4530; Fax: (713) 794-4901; E-mail: lji{at}mail.mdanderson.org
3 The abbreviations used are: TSG, tumor suppressor gene; NSCLC, non-small cell lung cancer; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; WT, wild type; HBEC, human bronchial epithelial cell; GFP, green fluorescence protein; pfu, plaque-forming unit(s); vp, viral particle; FACS, fluorescence-activated cell sorter; MOI, multiplicities of infection.
Received 1/ 4/01. Accepted 3/ 4/02.
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K.-H. Chun, H.-Y. Lee, K. Hassan, F. Khuri, W. K. Hong, and R. Lotan Implication of Protein Kinase B/Akt and Bcl-2/Bcl-XL Suppression by the Farnesyl Transferase Inhibitor SCH66336 in Apoptosis Induction in Squamous Carcinoma Cells Cancer Res., August 15, 2003; 63(16): 4796 - 4800. [Abstract] [Full Text] [PDF]
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 P. W. Schenk, M. Brok, A. W. M. Boersma, J. A. Brandsma, H. Den Dulk, H. Burger, G. Stoter, J. Brouwer, and K. Nooter Anticancer Drug Resistance Induced by Disruption of the Saccharomyces cerevisiae NPR2 Gene: a Novel Component Involved in Cisplatin- and Doxorubicin-Provoked Cell Kill Mol. Pharmacol., August 1, 2003; 64(2): 259 - 268. [Abstract] [Full Text] [PDF]
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 H.-Y. Lee, H. Srinivas, D. Xia, Y. Lu, R. Superty, R. LaPushin, C. Gomez-Manzano, A. M. Gal, G. L. Walsh, T. Force, et al. Evidence That Phosphatidylinositol 3-Kinase- and Mitogen-activated Protein Kinase Kinase-4/c-Jun NH2-terminal Kinase-dependent Pathways Cooperate to Maintain Lung Cancer Cell Survival J. Biol. Chem., June 20, 2003; 278(26): 23630 - 23638. [Abstract] [Full Text] [PDF]
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 Y.-H. J. Chow, A. Alberti, M. Mura, C. Pretto, P. Murcia, L. M. Albritton, and M. Palmarini Transformation of Rodent Fibroblasts by the Jaagsiekte Sheep Retrovirus Envelope Is Receptor Independent and Does Not Require the Surface Domain J. Virol., June 1, 2003; 77(11): 6341 - 6350. [Abstract] [Full Text] [PDF]
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M. Kochetkova, O. L. D. McKenzie, A. J. Bais, J. M. Martin, G. A. Secker, R. Seshadri, J. A. Powell, S. J. Hinze, A. E. Gardner, H. E. Spendlove, et al. CBFA2T3 (MTG16) Is a Putative Breast Tumor Suppressor Gene from the Breast Cancer Loss of Heterozygosity Region at 16q24.3 Cancer Res., August 15, 2002; 62(16): 4599 - 4604. [Abstract] [Full Text] [PDF]
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0.0001–0.005) but not significant in H358 and HBEC cell lines at both 3 and 5 days post-transduction (P 
