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Reversion of Transformed Phenotype in Ovarian Cancer Cells by Intracellular Expression of Anti Folate Receptor Antibodies

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Reversion of Transformed Phenotype in Ovarian Cancer Cells by Intracellular Expression of Anti Folate Receptor Antibodies Gene Therapy (2003) 10, 1018–1025 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies M Figini1, R Ferri1, D Mezzanzanica1, M Bagnoli1,2, E Luison1, S Miotti1 and S Canevari1 1Department of Experimental Oncology, Unit of Molecular Therapies, Istituto Nazionale Tumori, Milan, Italy; and 2Department of Experimental Medicine, University of L’Aquila, Italy The a-folate receptor (FR) is selectively overexpressed in typic cultures. The anti-FR scFv inhibited FR expression from 90% of nonmucinous ovarian carcinomas, whereas no 60 to 99%. At physiological concentrations of folate, expression is detectable in normal ovarian surface epithelium proliferation varied directly as a function of FR expression. (OSE). Indirect evidence suggests that FR expression is FR downmodulation was accompanied by reduced colony- associated with tumor progression and affects cell prolifera- forming ability in soft agar, morphological change of the cells, tion. To evaluate better the role of FR, we developed an significant enhanced adhesion to laminin or Matrigel, a two- approach based on intracellular expression of single-chain to three-fold increase in a6b4 integrin expression, and a (sc) antibodies (intrabody) to downmodulate membrane marked reduction in laminin production. In three-dimensional expression of FR in ovary cancer cells. IGROV-1 and organotypic cultures, anti-FR intrabody-transfected IGROV1 SKOV3 ovarian carcinoma cell lines were transfected with cells grew as a single-ordered layer, reminiscent of normal an anti-FR intrabody. Transfectants and parental cells were OSE growth in vivo. In conclusion, the anti-FR intrabody tested for FR, integrins and anti-FR intrabody expression by reverses the transformed phenotype in ovary cancer cells fluorescence-activated cell sorting (FACS), reverse tran- and may provide an efficient means to inhibit selectively the scription and polymerase chain reaction (RT-PCR) and/or growth of these cells. immunoblotting. Cell growth characteristics and adhesion Gene Therapy (2003) 10, 1018–1025. doi:10.1038/sj.gt.3301962 properties were evaluated in liquid, semisolid and organo- Keywords: intrabody; ovarian carcinoma; human a-folate receptor; organotypic culture; phenotype reversion Introduction a 38–40 kDa glycosyl-phosphatidyl-inositol-anchored mo- lecule10–13 that binds folic acid with high affinity. Detailed Genetic engineering of antibodies has opened new immunohistochemical analysis and evaluation of messen- avenues of therapeutic intervention1 and the possibility ger RNA expression revealed moderate levels of FR to examine in detail the pathophysiological role of some expression on the cell surface of normal epithelial cells of cellular/tumor markers. kidney, lung and breast, and high levels in placental The use of intrabodies targeted to the endoplasmic tissue.9,14–17 FR is not expressed in normal ovarian surface reticulum (ER) has recently emerged as a highly efficient epithelium (OSE)18 but is selectively overexpressed in the method to downregulate or ablate expression of a protein vast majority of nonmucinous ovarian carcinomas,9,13 normally secreted or expressed on the cell surface.2,3 The which arise from OSE. The association of FR expression stable introduction of a gene encoding intrabodies with progression of the disease13 has implicated this provides a powerful alternative to antisense RNA and molecule in malignant transformation of this tissue. We other methods of gene inactivation.3–5 The use of this recently showed that FR affects cell proliferation of method to functionally knockout proteins with a relevant ovarian carcinoma cells not only by mediating folate role in oncologic processes has given rise to various uptake but also by generating intracellular signals that are phenotypic effects in tumor cells, including alterations associated with dysregulated cell proliferation.19 We also consistent with the reversion of the malignant pheno- demonstrated that FR gene transcription is negatively type.5,6 Intrabodies are potentially suited for antiviral affected by the induction of the tumor suppressor gene and antitumor gene therapy approaches, and preclinical cav-1.18 Two monoclonal antibody/ies (Mab), MOV18 and and clinical studies have been promising.7,8 MOV19, directed to nonoverlapping epitopes of FR, have One of the more specific tumor markers identified in been generated in our laboratories.9 These Mab, and their our laboratory9 is the a-isoform of the folate receptor (FR), chimeric or bispecific derivatives, were entered in several diagnostic and therapeutic trials with some relevant 20,21 Correspondence: Dr S Canevari, Unit of Molecular Therapies, clinical responses. Instituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy To gain insight into the role of FR in ovarian cancer Received 18 June 2002; accepted 12 November 2002 progression, we exploited the specificity of the anti FR FR down-modulation in ovary cancer cells by intrabody M Figini et al 1019 a 48 48 48 wild type irrelevant mock 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 48 48 DM60 48 DM85 DM99 Relative cell number 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Fluorescence Intensity b c DM60 DM85 DM99 mock wild type wild type DM99 DM85 mock 100 100 94 60 DM60 Folate scFv receptor β-actin scFv Figure 1 Downmodulation of FR cell surface expression by anti-FR intrabody expression in IGROV1 ovarian carcinoma cells. (a) FACS analysis of three clones with an average downmodulation of the mean fluorescence intensity of 60 (DM60), 85 (DM85) and 99 (DM99)% compared to wild-type, mock- and irrelevant-transfected IGROV1 cells. Profiles are from one of five independent experiments with superimposable results. Filled curve: cells labeled with secondary antibody alone; thin line: cells labeled with Mab MOV18; thick line: cells labeled with Mab MOV19. (b) FR and scFv protein expression by Western blot analysis of whole-cell lysate from wild-type cells and anti-FR intrabody-transfected DM clones or mock transfectants. Results are froma representative experiment; Upper panel: FR expression, FR migrated at 38–40 kDa as a broad band because of extensive glycosylation; numbers indicate the percentage of FR levels relative to wild-type cells using b-actin as a loading control (not shown). Lower panel: scFv expression, scFv migrated at 30 kDa as a very faint band detectable only after long exposure and an ECL-plus development. (c) RT-PCR of intracellular scFv mRNA expression in wild-type cells and anti-FR intrabody-transfected DM clones or mock transfectants. Results are from one of three independent experiments with superimposable results. b- actin was used as a control for RNA quantity and integrity. MOV19 Mab to construct a single-chain (sc)Fv intrabody using single clones of transfected IGROV1 cells, obtained and tested the effects of functional downregulation of FR by limiting dilution, revealed a decrease in FR surface membrane expression on ovarian tumor cell proliferation expression ranging from 50 to 100%. Three clones were and adhesion. identified that showed an average decrease in the mean fluorescence intensity (MFI) of 60710 (DM60), 8575 (DM85) and 9971 (DM99)%, respectively, in five Results repeated different experiments. Figure 1a reports a representative experiment. By contrast, several different Downregulation of FR by anti-FR intrabody expression clones isolated by limiting dilution of IGROV1 wild-type Fluorescence-activated cell sorting (FACS) analysis of cells showed an MFI that varied less than 20% from that IGROV-1, SKOV3 and CHO cells stably transfected with of the original mixed population (data not shown). The the MOV19 scFv intrabody construct and selected in decrease in FR expression in intrabody-transfected cells G418-containing medium revealed heterogeneous ex- was evident not only at the cell surface, but also in the pression of FR on the IGROV1 cell surface, whereas whole-cell lysate as analyzed by Western blotting (Figure transfected SKOV3 and control transfected CHO cells 1b). In agreement with the FACS analysis, the FR protein were completely negative. Intact murine IgG Mab was undetectable in DM99, present in tiny amounts in MOV19 that recognizes the same epitope of scFv intra- DM85 and in larger amounts in DM60 as compared to body and MOV18 that recognizes a nonoverlapping the total FR protein produced by wild-type and mock epitope were used for FR detection. No decrease in FR cells. surface expression was detected in IGROV1 cells Reverse transcription and polymerase chain reaction transfected with mock vector or irrelevant scFv as (RT-PCR) analysis of intracellular scFv mRNA expres- compared to wild-type cells (Figure 1a). Similar analysis sion in IGROV1 cells showed amplification in the control Gene Therapy FR down-modulation in ovary cancer cells by intrabody M Figini et al 1020 ab150 2,3x103 nM folic acid 100 50 0 150 2-10 nM folic acid % of proliferation 100 50 0 mock mock DM60 DM85 DM99 DM60 DM85 DM99 wild type wild type irrelevant irrelevant Figure 2 Effects of anti-FR intrabody expression on cell growth of IGROV1 ovarian carcinoma cells. (a) Proliferation in liquid culture was assessed by cell counting based on trypan blue exclusion at 4 (open columns) or 7 (full columns) days after seeding in high (2.3  103 nM) (upper panel) or physiologic (2– 10 nM) (lower panel) folic acid concentrations. Data are given as mean (+s.d.) percent of wild-type cell proliferation of three replicates of one of three independent experiments. Asterisks indicate significant differences in proliferation as compared to that of wild-type cells by a two-tailed unpaired t-test. The correlation between FR downmodulation and proliferation inhibition at physiologic folic acid concentrations was evaluated using a two-tailed Pearson’s correlation with a 95% confidence interval. (b) Colony-forming ability of mock-transfected and DM60, DM85 and DM99 clones after 20 days of culture in soft agar (original magnification  40; scale bar represents 50 mm).
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