Immortalization of Syrian Hamster Embryo Cells: Probabilistic Event or Deterministic Process1

Previous findings on the induction of immortalization in SHE cells have been explained with the activation/alteration hypothesis which postulates that treatment with a carcinogen results in the induction of a so-called "activated state" which enhances the rate of a probabilistic event in the progeny of the treated cells. This event is supposed to be a mutation. Because it has been recently indicated that in mammalian cells the switch ing on of signal transduction pathways by 12-0-tetradecanoyI-phorbol13-acetate (TPA) or carcinogens can lead to genetic instability in the progeny of the treated cells, the possibility of an analogy between the induction of genetic instability and induction of immortalization after treatment with TPA was investigated. No effect of TPA was found on the rate of immortalization/cell/ generation, not in otherwise untreated cells nor in cells treated with benzo(a)pyrene. TPA was found to enhance the life span of SHE cells. The life span of a culture correlated with its growth rate and its cell density at confluence both in the absence and presence of TPA. These correlations are supposed to reflect a regulation mechanism involved in the program of cellular senescence, and supposedly TPA can partly reverse this program. Treatment with benzo(a)pyrene also interferes with the life span result ing in premature senescence in most of the cells and extension of life span in a small fraction of the cells which subsequently can become immortal. Repeated switching from logarithmic growth to G(l also enhanced life span and rate of immortalization. The findings indicate that the activated state is a disturbance of a differentiation program affecting in SHE cells the program of cellular senescence and that, as an explanation for immortalization, epigenetic alterations causing a deterministic process of dedifferentiation in a subpopulation of the cells appear as plausible or perhaps even more plausible as a probabilistic mutation. This indicates that disturbance of differentia tion might be among the causes of genetic instability. INTRODUCTION Previous studies on immortalization, using SHE3 cells as model system, revealed some unexpected features of this event. Firstly, im mortalization was not induced as a single event but appeared to be the result of a process consisting out of several sequential steps which occurred in a fixed sequence (1). Secondly, the induction of this process could not be due to the direct induction of a mutation (2). The results were interpreted as support for the probabilistic theory devel oped in 1980 by both Fernandez et al. (3) and Kennedy et al. (4) which states that treatment of C3H10t!4 cells with a carcinogen results in a so-called "activated state" of virtually all the treated cells which is transmitted to the progeny and which results in an enhanced rate of transforming events c.q. an enhanced rate of immortalization. Received 2/12/93; accepted 8/6/93. 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. 1This paper is the third in a series on immortalization. The research was supported by Ihe Dulch Cancer Foundation. Grant 1KW 87-4, by the J. A. Cohen Institute for Radiopathology and Radiation Protection, and by contract B-16-E-141-NL and F13P-CT920028 of the Association between the European Community with the University of Leiden. 2 To whom requests for reprints should be addressed. -'The abbreviations used are: SHE, Syrian hamster embryo; B(a)P, benzo(a)pyrene; TPA, ^-O-tetradecanoyl-phorbol-l.Vacetate; ELS, extended life span; PD, population doublings; PDT population doubling time; CD, cell density. The nature of the processes proposed by this activation/alteration hypothesis is entirely unknown. The induction of an activated state resembles the induction of SOS functions in bacterial cells and the secondary probabilistic alteration has been supposed to be a point mutation. SOS functions probably do not occur in mammalian cells, but recently it has been indicated that in mammalian cells switching on of signal transduction pathways by carcinogens or TPA can lead to ge netic instability with enhanced mutation rates (5, 6). Therefore, in this study it was investigated whether induction of immortalization re sembles induction of genetic instability. To answer this question, cells were treated with TPA. TPA is known to have an effect in the activation/alteration mecha nism of cell transformation as it enhances the rate of the transforma tion frequency of X-irradiated C3H10t!/2 cells (7). Moreover, TPA might enhance the frequency of immortalization of SHE cells as there is evidence that TPA enhances the frequency of morphological trans formation of SHE cells after treatment with carcinogen (8) and be cause morphological transformation of SHE cells predisposes for immortalization (2, 9). Surprisingly very few data appear available in the literature on the effect of tumor promoters on immortalization. It has been reported that continuous treatment with the tumor promoters TPA or 12-O-decanoyl-4a-phorbol-13-decanoate extends the life span of SHE cells twofold without inducing immortalization (10, 11). This extension has been ascribed to maintaining a proportion of the cells in a less differentiated state (11, 12). Although in these experiments, which used SHE cells from 13day-old embryos, no induction of immortalization was observed, a 4-5-fold enhancement of immortal ization was reported for SHE cells from 9-day-old embryos (13). Induction of immortalization by TPA has also been reported for cells derived from rat trachea! epithelium (14). Surprisingly no data appear available which relate to the effect of tumor promoters on carcinogen-induced immortalization. MATERIALS AND METHODS Cells and Culture Conditions. Syrian hamsters (Cpb-ShGa 51) in their second pregnancy were purchased from CPB-TNO, Zcist, The Netherlands. Preparation and cryopreservation of primary cultures was carried out as de scribed by Pienta et al. (15). All experiments were performed with tertiary cultures obtained from these cryopreserved stocks. Cells were grown in Duibecco's modified minimal essential medium supplemented with 20 or 5% fetal calf serum, NaHCOi (2.4 g/liter), and antibiotics at 37°Cin an atmosphere of 10% CO2 in a humidified incubator. Under these conditions the pH of the medium is 7.2. The cells were cultured in plastic Petri dishes or flasks (Greiner); in one experiment plastic Leighton tubes (Costar) with a surface of 5 cm2 were used on which the cells were growing equally well. The cultures were refed with fresh medium every 3 or 4 days. Cells were passaged by gentle trypsinization with 0.25% trypsin/EDTA solution for 5 min at 37°C. Treatment with Carcinogens and TPA. B(o)P was obtained from Sigma Chemical Co. It was dissolved in dimethyl sulfoxide and diluted lo the desired concentration into growth medium. Tertiary passage cultures were plated and allowed to attach overnight in normal medium. The cultures were rinsed with phosphate-buffered saline and treated with B(a)P (20 jug/ml) for 24 h. After treatment, the cultures were washed with phosphate-buffered saline. Control cultures were treated with solvent only (0.1% dimethyl sulfoxide). The treat-