Some biochemical characteristics of L1210 cell lines resistant to 6-mercaptopurine and 6-thioguanine and with increased sensitivity to methotrexate.

L1210 cells resistant to 6MP and 6TG exhibit increased sensitivity to MTX compared to the parent line. The differential response of parent and purine analog-resistant cell lines to MTX is not due to host influences, for both L1210/6MP and L1210/6TG cell lines are cross-resistant to 6-MeMPR, an inhibitor of de novo synthesis, and cultured L1210/6MP cells are more sensitive to MTX than the parent cell line. Following treatment of tumor-bearing mice with MTX, the drug concentration in L1210/6TG cells was about 50% greater than in L1210/0 cells for 24 hr and may account, wholly or in part, for the increased sensitivity of the L1210/6TG cell line to MTX. L1210/6MP cells, however, accumulated less MTX than L1210/0 cells, indicating that an equivalent mechanism is not operative in these cells. DHFR activity in L1210/6TG cells was the same as that in L1210/0 cells, but activity in L1210/6MP cells was lower by 60%. Cultured L1210/6MP cells also exhibited a deficiency in DHFR activity as compared to the parent cell line. The sensitivity of the enzyme to MTX was the same for all three cell lines propagated in vivo. Therefore, the increased sensitivity of the L1210/6MP cell line to MTX may be due, in part, to decreased DHFR activity. Significantly lower levels of GTP + GDP and CTP in 6TG-resistant cells than in parent cells 4 hr after the administration of MTX to tumor-bearing mice may be related to the increased MTX sensitivity of these cells. Our results indicate that the observed alterations in drug sensitivity are associated with more than one biochemical change and that these changes are different in the two purine analog-resistant cell lines.

The importance of short term exposure of C3H 10T1/2 cells to polycyclic hydrocarbons: evidence for hydrocarbon-mediated anticarcinogenic activity.

Since it has been shown that transformation frequencies (TF) of cultured mammalian cells exposed to polycyclic aromatic hydrocarbons (PAH) reach a maximum with increasing PAH concentration and then decline, we have examined TF in C3H 10T1/2 CL8 (10T1/2) cells as a function of an additional parameter of treatment, length of exposure. A 15-min exposure to either benzo[alpha]pyrene (BP) or 3-methylcholanthrene (3-MC), at concentrations ranging from 0.3 to 10 micrograms/ml, was sufficient to induce transformation suggesting that in 10T1/2 cells, non-induced enzymes of the cytochrome P-450 system are involved in the metabolic activation of PAH. At lower BP concentrations (0.3-1.25 micrograms/ml), TF generally increased with exposure time; at higher BP concentrations (2.5-10 micrograms/ml) maximal TF were achieved with 3 h of exposure. For 3-MC, maximal TF occurred at 0.5-1 h with a concentration of 10 micrograms/ml and at 1-6 h with lower concentrations. Moreover, low TF were obtained after 12-h and 24-h exposures to 10 micrograms/ml BP or 3-MC. These results show that TF depend on both the length of exposure and concentration of PAH. Since both BP and 3-MC are extensively metabolized to polyoxygenated derivatives and conjugates, we suggest that certain metabolites may be anticarcinogenic or antipromoting agents. The identities of such metabolites are yet to be determined.

Further evidence for an inhibitor of proliferation elaborated by normal human fibroblasts in culture: partial characterization of the inhibitor.

We present evidence that extends our earlier preliminary report on the stimulatory effect of saline washes on confluent cells in culture (Lacey et al., '77). That work suggested that an inhibitory substance was being removed by the washes. The present work suggests that the inhibitor is in the 10,000-30,000 MW range, is reversibly bound, is cationic and is also a protease inhibitor. It is heat stable, but is apparently degraded with time in our experimental systems.

Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T1/2 CL8 cell line.

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T1/2 CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent Km-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 microM BP, 3.3 nM NADPH) than in confluent cells (0.74-1.39 microM BP, 33.4-53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the Km-value for BP was less in log phase (0.2-0.4 microM BP) than in confluent cells (0.64-1.05 microM BP). Moreover, the Km-values for BP and NADPH in control cultures at confluency (0.10-0.14 microM BP, 15.4-23.2 nM NADPH) were less than those for BA-treated cells (0.64 microM BP, 37.9-54.8 nM NADPH) under the same nutritional conditions. The finding that the Km-values for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.

Establishment of mouse colonic carcinoma cell lines with different metastatic properties.

Tumorigenic cell lines were established in culture from three transplantable mouse colonic carcinomas designated CT 26, CT36, and CT 51. The cultured lines were characterized for the retention of the biological characteristics of the parental lines. All three cultured lines retained the ability to form tumors in vivo. Serially transplanted parental lines CT 26 and CT 51 grew at a faster rate than did CT 36 and showed a greater propensity for the formation of lung metastases. Similar characteristics were exhibited by the tumors formed from the injection of cultured cells. The cultured cell lines were also evaluated with respect to a number of in vitro markers for cancer. Cultured CT 26 and CT 51 cells formed tumors at lower inocula than did CT 36. CT 26 and CT 51 showed anchorage-independent growth and lack of contact inhibition, while CT 36 grew as a strict monolayer and did not form colonies in 0.27% agarose. CT 26 had the highest saturation density of the cell lines when grown in media supplemented with either 10 or 2.5% fetal bovine serum, while CT 51 had the lowest saturation density under these conditions. The varying degrees of malignancy exhibited by the three cell lines and the overall retention of the biological characteristics of the parental lines by the cultured lines suggest that the cultured cells (without the contaminating stromal elements present in the serially transplanted lines) will provide suitable material for the investigation of the molecular bases of these malignant characteristics.