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Isolation and Characterization of Complementary DNA Clones Corresponding to Genes Induced in Mouse Epidermis in Vivo by Tumor Promoters1

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Isolation and Characterization of Complementary DNA Clones Corresponding to Genes Induced in Mouse Epidermis in Vivo by Tumor Promoters1 [CANCER RESEARCH 50, 1626-1633. March I. 1990] Isolation and Characterization of Complementary DNA Clones Corresponding to Genes Induced in Mouse Epidermis in Vivo by Tumor Promoters1 Staffan Bohm,2 Anna Berghard, Carl Pereswetoff-Morath, and Rune Toftgard Karolimka Institute, Center for BioTechnology, NOVVM, S-I4I 52 Huddinge, Sweden ABSTRACT via the activation of PKC as well as a defect in the regulation of differentiation and/or proliferation in initiated keratinocytes Complementary DNA clones representing genes in SENCAR mouse are assumed to be key events for tumor development in epider epidermis, the expression of which is induced 4 h after one topical application of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate mis. Extensive in vitro studies in a variety of different cell types (TPA) were isolated. Of 56 isolated complementary DNA clones, 32 have resulted in the identification of several TPA-induced genes were identified to be identical to either metallothioneins (MT-I and Mi and different /raws-acting factors regulating their transcrip- ll) or endogenous retroviral like (VL30) sequences. In situ hybridization tional induction. One example is the induction of human me and analysis of mRNA levels in cell fractions separated by density tallothionein IIA,via enhanced AP-1 and AP-2 activity (12,13). gradient centrifugation revealed that Ml induction was restricted to Recent findings indicate that AP-1 consists of heterodimers of keratinocytes in the basal cell layer. Immunohistochemistry and time- oncoproteins belonging to thefos and /'//// gene families (14). In kinetic studies on mRNA levels in mouse epidermis showed that the order to understand mechanisms behind the ability of TPA to increase in MT and VL30 RNAs coincide in time with a TPA-induced transient block in basal cell proliferation (3-12 h after TPA treatment). induce cell kinetic and biological changes associated with tumor promotion it is important to analyze the effects of TPA on gene MT immunoreactivity and transcript levels had returned to control values at a time point (24 h after treatment) when epidermis is known to expression in mouse skin in vivo. Differences with regard to the hyperproliferate. Treatment with other types of tumor promoters showed cell type, physiological state of the cell (i.e., differentiation that MT-I and M 1-II mRNAs were coordinately induced and indicated state, cycling versus quiescent), and microenvironment (in vitro that in-l,2-dioctanoylglycerol, 12-O-retinoylphorbol-13-acetate, and versus intact tissue) are likely to contribute in determining the mezerein induced MT to a lesser degree than TPA. The calcium iono- gene program affected by TPA. The expressions of a limited phore A23187 induced mRNA levels for MTs as well as VL30. VL30 number of genes including ODC (15), c-fos, c-myc (16), transin and MT mRNA levels were not found to be elevated in epidermal tumors (17), /i-actin (18), transforming growth factor 0(19), osteopon- whereas the mRNA level corresponding to glyceraldehyde-3-phosphate tin (20), and certain keratin subunits (21) are known to be dehydrogenase was elevated in tumors and induced by TPA with time- induced by TPA in mouse epidermis. kinetics that correlate with a TPA-induced hyperproliferation. These complementary DNA clones provide useful tools in the study of the gene- In this report we utilized the technique of differential screen regulating effects of TPA in a target tissue relevant for tumor promotion. ing of cDNA libraries to isolate additional genes that are induced in mouse epidermis at an early time point (4 h) after a single topical application of TPA. We describe the identifica INTRODUCTION tion of two groups of genes among the isolated clones, the metallothioneins (MT-I and MT-II) and endogenous retroviral- TPA' was first identified as a potent tumor promoter in the like sequences belonging to the VL30 gene family. We also mouse skin model of multistage carcinogenesis (1, 2). In this describe their temporal pattern of TPA induction, expression model the progression of a normal cell to a benign tumor in skin tumors, induction in response to other types of tumor (papi! Ionia ) may be viewed as a clonal expansion of an epider promoters, and the localized MT expression in epidermis. mal cell initiated by some genetic change, such as activation of the Harvey ras (c-Ha-ras) protooncogene (3-5). Exactly how repeated treatments with TPA can give the initiated cell a MATERIALS AND METHODS proliferative advantage is not known. Normal keratinocytes proliferate in the basal cell layer of epidermis, migrate to the Animal Treatments and RNA Isolation. Female SENCAR mice, 6 to 9 weeks old, were purchased from HaríanSprague-Dawley, Indianap surface during differentiation, and form terminally differen olis, IN. The backs of the mice were shaved 48 h prior to topical tiated enucleated squamous cells that are eventually shed (6). treatment with 10 nmol of TPA (Pharmacia, Uppsala, Sweden). Other Topical application of TPA on mouse skin elicits a strong chemical agents used where RPA (LC Services, Woburn, MA), mezer pleiotropic response, including a transient inhibition of prolif ein (LC Services), 4-O-methyl-TPA (Pharmacia), calcium ionophore eration, followed by a hyperplastic response and inflammation A23187 (Sigma Chemical Co., St. Louis, MO), and sn-l,2-dioctanoyl- (7-10). In addition, a subpopulation of keratinocytes shows an glycerol (Sigma). All agents (concentrations are given in "Results") increase in the rate of terminal differentiation (11). were applied in 200 n\ of acetone. Control mice were treated with 200 Induction of an altered program of gene expression by TPA n\ of acetone only. Mice were killed by cervical dislocation. Papillomas were induced by initiation with 100 nmol of 7,12-dimethyl- Received 8/4/89; revised 11/8/89; accepted 11/28/89. benz(a)anthracene followed by promotion with TPA (3 nmol twice The costs of publication of this article were defrayed in part by the payment weekly) for 12 weeks. Carcinomas were derived from papillomas, in of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. duced as described above, but subsequently treated with 20 mg urethan 1The present study was supported by the Swedish Work Health Fund (Project ¡.p.once weekly to accelerate malignant conversion (22). Total RNA 88-0162). the Swedish Medical Research Council (Project B90-13X-07482-05), was isolated from epidermis (time points are given in "Results") and and by Centrala Försoksdjursnamden (Project 88-01). S. B. is a recipient of a from pools of papillomas or carcinomas as previously described (15). predoctoral fellowship from the Swedish Cancer Research Society. 2To whom requests for reprints should be addressed. Poly(A)+ RNA was isolated by one binding-elution cycle on oligodeox- 'The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; ythymidylate cellulose type T-2 (Collaborative Research, Inc., Lexing PKC. protein kinase C; MT. metallothionein; AP, activator protein; ODC. ton, MA). ornithine decarboxylase; RPA, 12-O-retinoylphorbol-13-acetate; GAPDH. gly- ccraldehyde-3-phosphate dehydrogenase; LTR, long terminal repeal; cDNA, com cDNA Library Construction. A cDNA library was constructed using plementary DNA; poly(A)* RNA. polyadenylated RNA; SSC, standard saline- XgtlO as the cloning vector essentially as described by Huynh et al. citrate (lx SSC is 0.15 M NaCI-0.015 M sodium citrate. pH 7.0). (23). The library was packaged in vitro using a commercially available 1626 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. TUMOR PROMOTER-INDUCED GENES extract system (Promega Biotec, Madison, WI). The resulting cDNA blotted to Hybond-N filter using a Minifold II apparatus (Schleicher & library contained 1 x IO6 independent clones that were stored and Schuell). Chromosomal DNA was isolated from mouse liver (24), screened without amplification. digested with EcoRl or AarnHI (Promega), electrophoresed through a Differential Probe. Poly(A)* RNA prepared from TPA-treated mouse 1% agarose gel, and blotted onto nylon membranes by alkaline transfer epidermis was used as template for synthesis of'2P-labeled cDNA. The (30). Southern and dot blots were hybridized and washed using the conditions used were the same as for the library' construction except same conditions as for Northern analysis. that the reaction mixture contained 10 MM[a-32P]dCTP, [a-32P]dGTP, DNA Sequencing and Analysis. cDNA inserts were separated from and [a-"P]dATP (Amersham Solna. Sweden; specific activity, 800 Ci/ XDNA by electrophoresis through 1% low melting agarose and sub- mmol). "P-labeled cDNA was freed from its template RNA by incu cloned into the £coRIsite of pGEM3Z (Promega) using methods bation in 0.3 M NaOH-10 mM EDTA (2 h, 37°C),neutralized (0.5 described by Struhl (31) and Escherichia coli JM109 as the host. volume 1 M acetic acid), and passed over a Sephadex G-50 M column Double-stranded DNA was used for dideoxy sequencing (T7 Sequenase; (Pharmacia). Single-stranded cDNA (500 ng) was hybridized to USB, Cleveland, OH) with [«-/Aio-15S]dATP(1000 Ci/mmol; NEN/ poly(A)* RNA (25 Mg)prepared from acetone-treated epidermis in 8 ¿il DuPont). Analysis of the DNA sequences was performed using the of 0.5 M sodium phosphate buffer (pH 7.5)-l mM EDTA-0.1% sodium Genetics Computer Group sequence analysis software package, version dodecyl sulfate, overlaid with paraffin oil, and incubated for 20 h at 5.3 (32). 68°C(R0t4500). After dilution in preheated (60°C)0.12 M phosphate In Situ Hybridization. Formalin-fixed skin tissue was embedded in buffer (pH 7.4), separation of single- and double-stranded nucleic acids paraffin, transversally sectioned at 6-7 Mm,and mounted onto micro was performed on a 1-ml (bed volume) hydroxylapatite (DNA grade.
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