Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro.

The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk and analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed. We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis.

Microgel immunoblotting of thymus and nuclear extracts by unidirectional diffusion.

A variant immunoblotting procedure is presented, starting from sodium dodecyl sulfate-containing ultra-thin microgels. The use of these gels allows efficient transfer of proteins to an immobilizing matrix such as nitrocellulose, by simple uni-directional diffusion, without loss of resolution. To this end an assembly was developed, keeping the microgel and the immobilizing matrix in continuous contact. To standardize the technique, two protein extracts (a classical rabbit thymus extract and an original autologous nuclear extract) were used, together with reference antinuclear antisera. The method is fast, easy to perform, and perfectly reproducible. For these reasons, the technique is very suitable for screening a patient's sera on a large scale.

Antiinvasive activity of estramustine on malignant MO4 mouse cells and on DU-145 human prostate carcinoma cells in vitro.

Estramustine (EM) is a conjugate of estradiol and nor-nitrogen mustard (nor-HN2), which is effective in the treatment of prostate cancer. We have compared the effect of EM with that of the known microtubule inhibitor vinblastine (VLB) on the following functions of malignant MO4 mouse cells and of DU-145 human prostate cancer cells in vitro: directional migration, invasion; and the organization and the assembly/disassembly equilibrium of microtubule complexes. The circular area covered by cells migrating from an aggregate explanted on a solid substrate was taken as an index of directional migration. Invasion was studied through confrontation of MO4 or DU-145 cells with fragments of embryonic chick heart in organ culture. Microtubules were investigated immunocytochemically and through immunodetection on protein blots. VLB and EM inhibited directional migration and invasion of MO4 and DU-145 cells in a dose-dependent manner; equimolar combinations of estradiol plus nor-nitrogen mustard did not mimic these effects. At anti-invasive concentrations VLB led to partial disassembly of microtubule complexes, whereas EM resulted in an abnormal pattern of microtubule complexes without alteration of the overall assembly/disassembly equilibrium. Combined treatment with VLB and EM resulted in an enhanced VLB effect, namely complete disassembly. In all tests DU-145 cells were more sensitive to both VLB and EM than were MO4 cells, and the effects were less reversible. The present experiments showed that EM shares an anti-invasive activity with other microtubule inhibitors.

The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting.

The technique of SDS-polyacrylamide gel electrophoresis and immunoblotting was used to study the evolution of the IgG antibody response to Toxoplasma gondii antigens in sequential sera of acutely infected patients. The results show that the IgG immunoblot pattern can be a useful marker for the stage of T. gondii infection. A 35-kD antigen elicited the first IgG response soon after exposure. During the course of infection additional bands appeared consecutively, following a constant sequence, to evolve to a late-stage-specific pattern with about 13-15 major bands. This pattern, which was reached at least 4 months after infection, was also found in the immunoblots of 28 patients with a chronic (latent) T. gondii infection.