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J Clin Invest. 1995 September; 96(3): 1535–1547.
PMCID: PMC185779

Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo.


Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo.

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  • Henderson GB. Folate-binding proteins. Annu Rev Nutr. 1990;10:319–335. [PubMed]
  • Anderson RG, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. [PubMed]
  • Antony AC. The biological chemistry of folate receptors. Blood. 1992 Jun 1;79(11):2807–2820. [PubMed]
  • Antony AC, Bruno E, Briddell RA, Brandt JE, Verma RS, Hoffman R. Effect of perturbation of specific folate receptors during in vitro erythropoiesis. J Clin Invest. 1987 Dec;80(6):1618–1623. [PMC free article] [PubMed]
  • Antony AC, Briddell RA, Brandt JE, Straneva JE, Verma RS, Miller ME, Kalasinski LA, Hoffman R. Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes. J Clin Invest. 1991 Jan;87(1):313–325. [PMC free article] [PubMed]
  • Matsue H, Rothberg KG, Takashima A, Kamen BA, Anderson RG, Lacey SW. Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6006–6009. [PubMed]
  • Luhrs CA, Raskin CA, Durbin R, Wu B, Sadasivan E, McAllister W, Rothenberg SP. Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium. J Clin Invest. 1992 Sep;90(3):840–847. [PMC free article] [PubMed]
  • Chung KN, Saikawa Y, Paik TH, Dixon KH, Mulligan T, Cowan KH, Elwood PC. Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates. J Clin Invest. 1993 Apr;91(4):1289–1294. [PMC free article] [PubMed]
  • Bottero F, Tomassetti A, Canevari S, Miotti S, Ménard S, Colnaghi MI. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res. 1993 Dec 1;53(23):5791–5796. [PubMed]
  • Kane MA, Elwood PC, Portillo RM, Antony AC, Najfeld V, Finley A, Waxman S, Kolhouse JF. Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J Clin Invest. 1988 May;81(5):1398–1406. [PMC free article] [PubMed]
  • Mulligan RC. The basic science of gene therapy. Science. 1993 May 14;260(5110):926–932. [PubMed]
  • Carter BJ. Adeno-associated virus vectors. Curr Opin Biotechnol. 1992 Oct;3(5):533–539. [PubMed]
  • Berns KI. Parvovirus replication. Microbiol Rev. 1990 Sep;54(3):316–329. [PMC free article] [PubMed]
  • Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. [PubMed]
  • Samulski RJ. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. [PubMed]
  • Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. [PubMed]
  • Kotin RM, Menninger JC, Ward DC, Berns KI. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. [PubMed]
  • Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. [PubMed]
  • Walz C, Schlehofer JR. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17. J Virol. 1992 May;66(5):2990–3002. [PMC free article] [PubMed]
  • Zhou SZ, Broxmeyer HE, Cooper S, Harrington MA, Srivastava A. Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. Exp Hematol. 1993 Jul;21(7):928–933. [PubMed]
  • Flotte TR, Afione SA, Conrad C, McGrath SA, Solow R, Oka H, Zeitlin PL, Guggino WB, Carter BJ. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10613–10617. [PubMed]
  • Walsh CE, Liu JM, Xiao X, Young NS, Nienhuis AW, Samulski RJ. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7257–7261. [PubMed]
  • Elwood PC. Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells. J Biol Chem. 1989 Sep 5;264(25):14893–14901. [PubMed]
  • Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. [PMC free article] [PubMed]
  • McLaughlin SK, Collis P, Hermonat PL, Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6):1963–1973. [PMC free article] [PubMed]
  • Nahreini P, Srivastava A. Rescue and replication of the adeno-associated virus 2 genome in mortal and immortal human cells. Intervirology. 1989;30(2):74–85. [PubMed]
  • McHugh M, Cheng YC. Demonstration of a high affinity folate binder in human cell membranes and its characterization in cultured human KB cells. J Biol Chem. 1979 Nov 25;254(22):11312–11318. [PubMed]
  • Antony AC, Kane MA, Portillo RM, Elwood PC, Kolhouse JF. Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells. J Biol Chem. 1985 Dec 5;260(28):14911–14917. [PubMed]
  • Lavappa KS, Macy ML, Shannon JE. Examination of ATCC stocks for HeLa marker chromosomes in human cell lines. Nature. 1976 Jan 22;259(5540):211–213. [PubMed]
  • Lavappa KS. Survey of ATCC stocks of human cell lines for HeLa contamination. In Vitro. 1978 May;14(5):469–475. [PubMed]
  • Verma RS, Gullapalli S, Antony AC. Evidence that the hydrophobicity of isolated, in situ, and de novo-synthesized native human placental folate receptors is a function of glycosyl-phosphatidylinositol anchoring to membranes. J Biol Chem. 1992 Feb 25;267(6):4119–4127. [PubMed]
  • Antony AC, Kincade RS, Verma RS, Krishnan SR. Identification of high affinity folate binding proteins in human erythrocyte membranes. J Clin Invest. 1987 Sep;80(3):711–723. [PMC free article] [PubMed]
  • Antony AC, Utley C, Van Horne KC, Kolhouse JF. Isolation and characterization of a folate receptor from human placenta. J Biol Chem. 1981 Sep 25;256(18):9684–9692. [PubMed]
  • Srivastava CH, Samulski RJ, Lu L, Larsen SH, Srivastava A. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8078–8082. [PubMed]
  • McCutchan JH, Pagano JS. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Deutsch JC, Elwood PC, Portillo RM, Macey MG, Kolhouse JF. Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells. Arch Biochem Biophys. 1989 Nov 1;274(2):327–337. [PubMed]
  • Verma RS, Antony AC. Kinetic analysis, isolation, and characterization of hydrophilic folate-binding proteins released from chorionic villi cultured under serum-free conditions. J Biol Chem. 1991 Jul 5;266(19):12522–12535. [PubMed]
  • Zhen W, Jayaram HN, Weber G. Antitumor activity of tiazofurin in human colon carcinoma HT-29. Cancer Invest. 1992;10(6):505–511. [PubMed]
  • Elwood PC, Kane MA, Portillo RM, Kolhouse JF. The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells. J Biol Chem. 1986 Nov 25;261(33):15416–15423. [PubMed]
  • Knecht DA, Loomis WF. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. [PubMed]
  • Yokoyama K, Imamoto F. Transcriptional control of the endogenous MYC protooncogene by antisense RNA. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7363–7367. [PubMed]
  • Inouye M. Antisense RNA: its functions and applications in gene regulation--a review. Gene. 1988 Dec 10;72(1-2):25–34. [PubMed]
  • Antony AC, Kane MA, Krishnan SR, Kincade RS, Verma RS. Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins. Biochem J. 1989 Jun 1;260(2):401–411. [PubMed]
  • Anderson RG. Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr Opin Cell Biol. 1993 Aug;5(4):647–652. [PubMed]
  • Kane MA, Portillo RM, Elwood PC, Antony AC, Kolhouse JF. The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein. J Biol Chem. 1986 Jan 5;261(1):44–49. [PubMed]
  • Elwood PC, Deutsch JC, Kolhouse JF. The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease. J Biol Chem. 1991 Feb 5;266(4):2346–2353. [PubMed]
  • Luhrs CA, Slomiany BL. A human membrane-associated folate binding protein is anchored by a glycosyl-phosphatidylinositol tail. J Biol Chem. 1989 Dec 25;264(36):21446–21449. [PubMed]
  • Leamon CP, Low PS. Cytotoxicity of momordin-folate conjugates in cultured human cells. J Biol Chem. 1992 Dec 15;267(35):24966–24971. [PubMed]
  • Leamon CP, Pastan I, Low PS. Cytotoxicity of folate-Pseudomonas exotoxin conjugates toward tumor cells. Contribution of translocation domain. J Biol Chem. 1993 Nov 25;268(33):24847–24854. [PubMed]
  • Lee RJ, Low PS. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis. J Biol Chem. 1994 Feb 4;269(5):3198–3204. [PubMed]

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