3T3 cells

Luis Roderiguez Fernandez, J., Geiger, B., Salmon, D., Ben-Ze ev, A. (1993). Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells. J. Cell Biol. 122, 1285-1294. 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Inhibition of EGF binding by palytoxin could be due to a decrease in receptor affinity, as in the case of TPA-type tumor promoters, and/or a decrease in receptor number. In Swiss 3T3 cells there are two classes of EGF receptors. The dissociation constants for the two EGF receptor classes were determined to be approximately 2 X 10 M and 2 x 10" M, corresponding to approximately 1 x 10 and 1 X 10 receptor molecules per cell, respectively (33). Scatchard analysis revealed that treatment of Swiss 3T3 cells with palytoxin, like PDBu, caused an apparent loss in high-affinity binding (Figure 2). However, in contrast to PDBu, palytoxin also caused a significant (approximately 50%) loss of low affinity EGF binding. 

The differences between palytoxin and PDBu with respect to kinetics, temperature dependence, and effect on low affinity binding suggest that these two different types of tumor promoters may be acting through different mechanisms. Further, in contrast to PDBu, the effect of palytoxin is not readily reversible (33). To determine where the two pathways differ, we compared the relative ability of palytoxin and PDBu to inhibit EGF binding in protein kinase C depleted cells. Swiss 3T3 cells were depleted of protein kinase C to different extents by exposing confluent quiescent cells to 0, 20, 200, or 2000 nM PDBu for 72 hr. Previous results indicate that this treatment depletes cells of protein kinase C activity in a dose-dependent manner (31). 

Figure 1. Effect of tumor promoter treatment on binding of I-EGF to Swiss 3T3 cells. Confluent quiescent Swiss 3T3 cells (A and B) were treated for the indicated times at 37 C with PDBu [A 2 nM (o), 20 nM (A), 200 nM ( )] or palytoxin [B 1.1 pM ( ), 3.7 pM...

Table I. Effect of Palytoxin on DNA Synthesis in Swiss 3T3 Cells...

Confluent Swiss 3T3 cells were serum-starved by incubation for 48 hr in DME containing 0.1% PCS. Cells were then incubated with the indicated compound at 37 C in the presence of H-thymidine, 0.1 or 10% PCS for 19.5 hr, washed, and then assayed for H-thymidine incorporation into DNA as described in Methods. 

Figure 2. Scatchard analysis of I-EGF binding to Swiss 3T3 cells treated with PDBu or palytoxin. Confluent quiescent Swiss 3T3 cells were treated at 37 C with solvent (o) or 200 nM PDBu ( ) for 15 min (Upper panel) or with solvent (o) or 11 pM palytoxin ( ) for 60 min (Lower panel). Cells were assayed as in Figure 1. (Reproduced with permission from Ref. 33. Copyright 1987 Cancer Research, Inc.)...

Na also appears to play a role in palytoxin action in some systems. To determine if there is a Na requirement for palytoxin action on the EGF receptor, Swiss 3T3 cells were assayed for palytoxin activity in Na containing medium versus Na deficient medium. When NaCl is replac by cholineCl, palytoxin can no longer inhibit EGF binding in Swiss 3T3 cells (Figure 5). By contrast, PDBu is equipotent in both Na containing and Na free media (data not shown). 

Because these results suggest that extracellular Na is required for inhibition of EGF binding by palytoxin in these cells, we determined if palytoxin caused Na influx in Swiss 3T3 cells. When Na influx was monitored at an early time point (7 min), it was found that palytoxin causes an influx of Na and that the rate of Na influx is dose dependent (Figure 6). In parallel with its effect on EGF binding, palytoxin at different doses increases intracellular Na to the same final level (42). Although Na influx occurs prior to the inhibition of EGF binding, these results and the apparent Na dependence of the palytoxin effect suggest a role for Na in the action of palytoxin on the EGF receptor. 

Figure 4. Effect of Ca on palytoxin action in Swiss 3T3 cells. Confluent quiescent Swiss 3T3 cells were incubated for the indicated times at 37 C with Ca -free incubation media containing 100 pM EGTA (4), complete incubation media containing 3.7 pM PTX (0), or Ca -free incubation media containing 100 pM EGTA plus 3.7 pM PTX Cells were assayed as in Figure 1. Data expressed as percentage of I-EGF binding in cells treated with complete incubation media alone.

Figure 6. Effect of palytoxin on the rate of Na influx in Swiss 3T3 cells. Confluent quiescent Swiss 3T3 cells were incubated for 37 C for 7 min in incubation media containing 0.1 pM PTX, 1.1 pM PTX, or 11 pM PTX. Intracellular Na was determined as described in the Experimental section. Data points represent the mean of quadruplicate points.

Tu AS, Murray TA, Hatch KM, et al. 1985. In vitro transformation of BALB/C-3T3 cells by chlorinated ethanes and ethylenes. Cancer Lett 28 85-92. 

LiNASSiER c, PIERRE M, LE PECQ J B and PIERRE J (1990) Mechanism of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity. Biochem Pharmacol. 39 (1) 187-93. 

Weitzman, S., Schmeichel, C., Turk, P., Stevens, C., Tolsma, S. and Bouck, N. (1988). Phagocyte-mediated carcinogenesis DNA from phagocyte-transformed C3H lOTl/2 cells can transform NIH/3T3 cells. Ann. N. York Acad. Sci. 551, 103-109. 

K. Luby-Phelps, P. E. Castle, D. L. Taylor, and F. Lanni, Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells, Proc. Natl. Acad. Sci. USA 84, 4910 (1987). 

Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990). Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. J. Biol. Chem. 265 11487-11494. 

Figure 6. The c-mos negative regulatory element (NRE). Nucleotide positions of the NRE are shown relative to the spermatocyte transcription start site, taken as 280 base pairs upstream of the c-mos ATG (see Fig. 4). The endpoints of the NRE are defined by deletions that allow c-mos expression in NIH 3T3 and other somatic cells. Mutations of the sequences designated by boxes 1,2, and 3 also allow c-mos transcription in NIH 3T3 cells, indicating that these sequences represent functional elements within the NRE. Boxes 1 and 2 are similar to sequences upstream of the protamine (Prot) promoter that inhibit in vitro transcription in HeLa cell extracts. A sequence just upstream of box 2 is also similar to a putative repressor-binding site in the regulatory region of Pgk2.

It should be noted that the NRE defined in these experiments is distinct from the previously described c-mos UMS sequence (Blair et al., 1984 Wood et al., 1984). The UMS is located approximately 1.4 kb upstream of the c-mos spermatocyte promoter and was identified because it blocked activation of c-mos transforming potential by insertion of retroviral promoters. It is thought to act as a transcriptional terminator, blocking transcription of c-mos initiated at upstream sequences. However, both the spermatocyte and oocyte transcription initiation sites are substantially downstream of the UMS. Moreover, the presence or absence of the UMS does not affect c-mos expression in either microin-jected oocytes (Pal et al., 1991) or transfected NIH 3T3 cells (Zinkel et al., 1992). It thus appears unlikely that the UMS functions as a negative regulator of c-mos transcription from either the spermatocyte or oocyte promoters in somatic cells. 

Cowley, S., Paterson, H., Kemp, R, and Marshall, C J. 1994. Activation of MAP kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77 841-52. 

Perocco, P., Paolini, M. Mazzullo, M. Biagi, GL., and Cantelli-Forti, G. 1999. Beta-carotene as enhancer of cell transforming activity of powerful carcinogens and cigarette-smoke condensate on BALB/c 3T3 cells in vitro. Mutat Res 440 83-90. 

Fig. 5 BALB/c 3T3 cell transformation assay example of focus induced in BALB/c 3T3 clone A31-1-1 after exposure to a carcinogenic compound. Cells are stained with Giemsa stain...

Fig. 6 BALB/c 3T3 cell transformation assay Colony formation assay...

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