in Human Embryonal Carcinoma Cells Vivo in and in Vitro-Diamminedichloroplatinum ( II ) Resistance cis Updated

In the embryonal carcinoma cell line Tera and its 3.7-fold cis-diamminedichloroplatinum(II) (CDDP)-resistant subline, Tera-CP, parameters were studied that might have changed in relation to induction of CDDP resistance. Phenotypes of both lines were embryonal carcinoma. Karyotypes were related with a decreased mean number of chromosomes and fewer copies of the short arm of chromosome 12 in Tera-CP. Tera-CP showed cross-resistance for melphalan and 4-hydroperoxycyclophosphamide and had an 1.4-fold increased glutathione (GSH) level, a 1.5-fold increased glutathione S-transferase (GST) activity, and a 1.4-fold increased GST~r expression compared to Tera. Tera-CP was cross-resistant to 5-fluorouracil, but thymidylate synthase activity was not increased. Topoisomerase I and II activities and c-myc RNA and protein expression were the same in both lines. Platinum accumulation was equal in both lines, and platinum-DNA binding was lower in Tera-CP than in Tera. Both cell lines were xenografted into nude mice and tumors showed marked differentiation. Tera-CP tumors were 2.8-fold resistant to CDDP compared to Tera tumors. In new cell lines derived from xenografts of Tera and Tera-CP CDDP sensitivity, GST activity and GSH level corresponded with their sensitivity and resistant origin. Tera-CP is a model of in vitro and in vivo CDDP resistance with the GSH/GST detoxifying system as an important mechanism. CDDP resistance could be induced without a concomitant increase in differentiation. I N T R O D U C T I O N Teratocarcinoma, a variant of germ cell tumor of the testis, is the most common form of cancer in young adult men (1). It consists of two histological components, namely EC 3 and teratoma (1). EC cells are the stem cells from which the teratoma component is derived (2). Clinically, EC is highly malignant with a tendency to early metastatic spread but is also a good responder to CDDP-containing chemotherapy (3). In vitro, EC cell lines are known to be more sensitive to CDDP than their more differentiated counterparts (4). Patients with teratocarcinomas treated with CDDP-containing chemotherapy regimens have cure rates of 70-80% (5). However, in the remaining patients, the tumor becomes untreatable due to development of drug resistance. Resistance to CDDP can be due to a variety of mechanisms. These include reduced drug accumulation and increased detoxification of CDDP in the cellular cytoplasm. In the cell nucleus, decreased DNA accessibility and increased DNA repair may play a role (6, 7). The Received 5/21/93; accepted 9/23/93. 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 indicate this fact. 1 Supported by Grants GUKC 88-11 and 90-18 from the Dutch Cancer Foundation. 2 To whom requests for reprints should be addressed, at Division of Medical Oncology, Department of Internal Medicine, University Hospital, Oostersingel 59, 9713 EZ Groningen, the Netherlands. 3 The abbreviations used are: EC, embryonal carcinoma; CDDP, cis-diamminedichloroplatinum(II) (cisplatin); CK, cytokeratin; 5-FUra, 5-fluorouracil; 4-HPC, 4-hydroperoxycyclophosphamide; GFAP, glial fibrillary acidic protein; IDso, the concentration of a drug reducing cell survival by 50%; RF, resistance factor (IDso Tera-CP:IDso Tera ratio); GSH, glutathione; TSH, total sulfhydryl groups; GST, glutathione S-transferase; FdUMP, 5-fluoro-2'-deoxyuridine 5'-monophosphate; AAS, atomic absorbance spectrophotometry; i(12p), isochromosome of the short arm of chromosome 12; TeraX, Tera xenograft; Tera-CPX, Tera-CP xenograft.CDNB, 1-chloro-2, 4-dinitrobenzene. latter is accompanied by increased amounts of repair enzymes (7, 8). Also, changes in the qTP synthesis might be an indication for increased DNA repair as this process requires a source of deoxynucleotides (8). The net effect of all these systems is reduced Pt-DNA and thus decreased cytotoxicity since the Pt-DNA interactions are considered to be the main cytotoxic lesions induced by CDDP (9). However, mechanisms of CDDP resistance are not uniform and vary among different cell lines. In this study we describe a CDDP-resistant subline (Tera-CP) of NT2/D1 (Tera; Ref. 10), an EC cell line with the capacity to differentiate in vitro. This model enabled us to study not only mechanisms underlying CDDP resistance in extremely sensitive ceils but also the effect of serial CDDP incubations on the EC phenotype of the cells. In addition, Tera cells in a xenografl in nude mice show marked differentiation (10), while in contrast, Tera in vitro is mainly composed of EC cells. To investigate the effects of this differentiation on CDDP resistance, Tera and Tera-CP were xenografted into nude mice, and tumor sensitivity for CDDP was determined. In new cell lines derived from the CDDP-treated sensitive and resistant xenografts, parameters thought to be relevant for resistance in the primary cell lines were measured in order to confirm their role in CDDP resistance. MATERIALS A N D M E T H O D S