Establishment and characterization of immortalized non-transplantable mouse mammary cell lines cloned from a MMTV-induced tumor cell line cultured for a long duration.

During the culturing of a mouse mammary tumor cell line, MuMT73, maintained in vitro for more than a decade, we found morphological heterogeneity in its cells; some showed contact inhibition in their growth, some formed domes and some grew criss cross and piled up. In trying to clone the cell line to isolate cells showing contact inhibition or dome formation, we were able to establish six clonal cell lines. These six cell lines were categorically divided into three groups according to their phenotypical behavior, Groups A, B and C. Group A (clones 1, 5 and 7) cells had a property of contact inhibition. They induced no tumor when injected into the subcutaneous tissue of the back, nor even when injected into the mammary fat pads or under the kidney capsule of syngenic or nude mice, and therefore were thought to be non-malignant in nature. They were positively stained by anti-keratin antiserum and had mouse mammary tumor viruses (MMTVs). Group B (clone 6) cells grew in a crisscross pattern and piled up, and they induced tumors when injected into the subcutaneous tissue of the back of mice. Group C (clones 3 and 4) cells formed domes in their growth and induced some tumors in the mammary fat pads and under the kidney capsule of KSN nude mice. In Southern blots with MMTV-env probe, numerous exogenous MMTV proviruses were detected in these cell lines. The insertion patterns of these proviruses in cells of non-malignant clonal lines (Group A) resembled those of malignant lines (Group B), except one band (about 26 Kb), but were considerably different from those of intermediate lines (Group C). On the other hand, no difference was detected in Southern blots with int-1 or int-2 probes among the non-malignant, intermediate and malignant clonal cell lines.