In situ microassay of ornithine decarboxylase.

Ornithine decarboxylase catalyses the principal step in the biosynthesis of polyamines (1, 2) and is reported to be a proto-oncogen implicated in the control of both FIG. 1. Modified rubber stopper. cell growth and transformation (3). Its activity is induced by a number of agents such as amino acids, pharmaceuticals, hormones, and growth stimuli (4). The cient to completely cover the plated cells so that very non-in situ assay methods of ornithine decarboxylase little radioactive substrate was needed. The screw-cap mentioned in the literature have several disadvanof each flask was removed and immediately replaced by tages: they require large numbers of cells, and the enzya modified rubber stopper. Figure 1 shows a perforated matic activity measured in cell-free extracts does not rubber stopper that contains an Eppendorf tube pushed necessarily reflect its true activity because the enzyme tightly into the hole to give a force-fit; the tube is 5.0 is unstable. The ornithine decarboxylase has a PEST mm diameter and 45 mm long, and its volume is 500 region (5) and is rapidly degraded. ml. The bottom of the tube was cut off with a razor Recently, a microassay of decarboxylation reactions blade 5 mm from the end. A hyamine-moistened paper in cultured cells was described by Bartos et al. (6). This strip (30 1 2 mm) was previously introduced into the method uses standard petri dishes with a modified tube to trap the CO2. At the desired incubation time, cover to permit the collection of CO2 evolved in the the reaction was stopped by injecting 0.5 ml of 50% decarboxylase reactions. We propose an alternative HClO4 with a hypodermic syringe through the rubber and simple method for the in situ determination of ornistopper. After 30 min the paper strip moistened with thine decarboxylase activity in cells cultured in ordihyamine from the Eppendorf tube was introduced into nary T-flasks. The only manipulation required for the the liquid scintillation counter vials and the CO2 proassay is replacing the screw-caps with modified rubber duced by the ornithine decarboxylase reaction was stoppers. counted by a Rack Beta (LKB, Sweden) scintillation We tested the method by using a new cell line (named CT-2) cloned in our laboratory from cells derived from a human colorectal carcinoma. The cells were cultured at 377C under 5% CO2 in DMEM–Ham’s F-12 culture medium that was supplemented with 10% FCS and antibiotics. The karyotypes of this newly established cell line determined after more than 50 metaphase analyses were 30% diploid and 55% hypodiploid (43– 44 chromosomes), and the remaining 15% were hyperdiploid, either triploid or tetraploid. Many micronuclei were observed. We also cultured the HT-29 human colon cell line from the ATCC to compare the results. The cells were seeded in ordinary cell-culture T-flasks, cell density was 5 1 10 cells/25 cm. To begin the test, the growth medium was discarded and replaced by 1.5 ml of PSS (1.2 mM KH2PO4, 16 mM NaH2PO4, 120 mM NaCl, 0.6 mM MgSO4, 3 mM KCl, 1.4 mM CaCl2, pH 7.4) containing 11 mM glucose, 1.0 mM L-ornithine and 5 mM [1-C]ornithine (0.4 mCi). This volume was suffi-