Effects of ProcoHagen C-Proteinase Enhancer Protein on the Growth of Cultured Rat Fibroblasts Revealed by an Excisable Retroviral Vector1

An excisable retroviral vector, TSN-Iox, was developed by exploiting Cre-IoxP homologous recombination. An integrated TSN-lox provirus could be excised, leaving a solo long terminal repeat at the integration site; inverse PCR, taking advantage of the solo long terminal repeat, was used to characterize cellular flanking sequences. A TSN-lox-transduced Rat2 cell clone, lox-7, was found to harbor the provirus in an intron of the procollagen C-proteinase enhancer protein (PCPE) gene, whose expression was lowered compared with that of the parental Rat2. When the vector provirus in Iox-7 cells was excised, PCPE expression was elevated. The level of PCPE expression seemed to affect cell growth properties such as morphology, contact inhibition, and anchorage-independent growth. These results suggested that the excisable retroviral vector may be useful for studying the molecular basis for proviral insertion mutagenesis, and that POPE may play a significant role in controlling cell growth and differentiation. Introduction Retrovirus has been shown to affect cellular gene expression through proviral integration into host chromosomes. For example, it has been demonstrated in a variety of animal systems that proviral integration within or in the vicinity of cellular proto-oncogenes or tumor suppressor genes could cause malignant disease (reviewed in Refs. i and 2). Retroviral insertion mutagenesis has also been used for studying the function of various other cellular genes (3-8). For studying the molecular mechanism of insertion mutagenesis, it is necessary to identify the proviral integration site by determining the chromosomal sequences flanking the proviral integration site. In addition, it would be valuable if cellular gene expression inactivated by proviral insertion could be restored in a controlled manner. Therefore, a retroviral vector system that facilitates these procedures would be useful. In this study, we constructed an excisable retroviral vector designated TSN-lox by adopting the Cre-/oxP-mediated homologous recombination of bacteriophage P1 (9, 10). After transduction into the rat fibroblast cell line Rat2, the integrated provirus of the TSN-lox vector could be excised by the transfection of a Cre protein expression plasmid, leaving a solo LTR3 behind. Taking advantage of this solo LW, cellular genomic sequences flanking the upstream and the downstream of the proviral integration site were simultaneously isolated by inverse PCR. Sequence analysis of the cellular sequences flanking the proviral integration sites revealed that among TSN-Iox-transduced Rat2 cell clones, Iox-7 harbored the provirus in an intron of the PCPE gene (1 i). Compared with parental Rat2 cells, lox-7 cells expressed a lower level of PCPE mRNA and protein, due to the inactivation of one of two PCPE alleles by proviral insertion. When the TSN-lox provirus in lox-7 cells was excised by Cre-/oxP-mediated homologous recombination, leaving a solo LW, PCPE expression was up-regulated, indicating that the allele inactivated by proviral insertion had been turned on again. Interestingly, the level of PCPE expression seemed to affect cell growth properties such as morphology, susceptibility to contact inhibition, and ability to grow in soft agar in an anchorage-independent manner. Thus, active excision of the proviral sequence integrated in the PCPE intron was associated with the induction of phenotypic reversion. These results suggest that the excisable retroviral vector may be useful for studying the effects of proviral insertion mutagenesis on host cell functions, and that PCPE may play a significant role in controlling cell growth and differentiation. Results Transduction of TSN-Iox Vector and Its Cre-mediated Excision in Rat2 Fibroblasts. The TSN-lox vector carries the HSV-i tk gene and the bacterial nec gene under the control of the 5’ LTR and the SV4O promoter, respectively Received 1/26/98; revised 4/7/98; accepted 4/7/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 Supported in part by research grantsfrom the Bureau of Social Security, the Japanese Ministry of Human Health and Welfare. 2 To whom requests for reprints should be addressed, at Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-i Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3812-2111, extension 3409; Fax: 81-3-5684-9374; E-mail: mmasuda0m.u-tokyo. ac.jp. 3 The abbreviations used are: LTR, long terminal repeat; BMP-1, bone morphogenic protein 1; BVDU, (E)-5-(2-bromovinyl)-2’-deoxyuridlne; EST, expressed sequence tag; HAT, hypoxanthine-aminoptenn-thymidine; HSV-1, herpes simplex virus type 1; MMLV, Moloney murine leukemia virus; PCPE, procollagen C-proteinase enhancer protein; PVDF, polyvinylidene difluoride; RACE, rapid amplification of cDNA ends; RSV, Rous sarcoma virus; Uc, thymidine kinase; 0418r, G418-resistant ( defined similarly for other terms throughout article); G418 , G418-sensitive (S defined similarly for other terms throughout article); GSP, gene-specific primer; DIG, digoxigenmn.