Pituitary extract causes aggregation and differentiation of rat mammary tumor MTW9/Pl cells

Abstract

Alkaline pituitary extracts (PE) of the mammosomatotropic tumor MtTW10 differentiated cultured PRL-independent rat mammary tumor cells MTW9/Pl. Within 24 h, MTW9 cells growing in suspension began to aggregate and adhere to the plastic dish. Cultured dispersed MTW9 cells were undifferentiated, but PE treatment resulted in organoid aggregates that exhibited glandular luminal structures and periodic acid-Schiff-positive material consistent with basement membrane formation. Morphometric examination of organoids demonstrated a reduction in nuclear and cell perimeters compared to those of untreated cells. Electron microscopy showed that the treatment resulted in polarization, nuclear changes, junctional complexes, secretory spaces, microvilli, and basement membrane formation. A disaggregated undifferentiated tumor now appeared as a differentiated adenocarcinoma. PE induced expression of laminin and milk protein, but failed to increase fibronectin expression. Extracts of MTOM (a variant of MtTW10 which secretes little PRL) and bovine pituitary also produced aggregation and adhesion of MTW9. A number of tissue extracts, growth factors, and hormones failed to produce such aggregation. Laminin, but not fibronectin, produced aggregation and adhesion similar to those produced by PE. Cycloheximide inhibited the aggregation effect of PE, but not that of laminin. PE was mitogenic for MTW9, but inhibition of proliferation by vinblastine did not inhibit the aggregation induced by PE. These observations suggest that the pituitary contains a novel factor that stimulates matrix synthesis, resulting in differentiation, possibly laminin induced.

A pituitary gene encodes a protein that produces differentiation of breast and prostate cancer cells

“…To isolate the tumor differentiation factor we used expression cloning in Xenopus oocytes (14, 15) and a biological assay based on the aggregating and differentiating effect of the PTE on breast carcinoma cells in culture (6). To determine whether this assay was sensitive enough to detect the protein encoded by the TDF cDNA clone isolated from a human pituitary adenoma library, we first determined the effect of the lysate prepared from oocytes injected with mRNA from a human pituitary tumor on MCF7 cells.…”

“…Preparation of cellular lysates, SDS͞PAGE, and transfer to nitrocellulose paper were performed as described (6). The blots were immunoblotted with anti-P1 Ab (Covance, Denver, PA) with a kit and the instructions from Pierce.…”

“…The proliferation assay was performed as described (6). In brief, cultures containing 1 ϫ 10 5 cells in 1 ml of serum-free medium were incubated in the presence of various concentrations of peptides (1 ng͞ml to 50 g͞ml) or recombinant TDF protein (rTDF; 1 ng͞ml to 50 g͞ml) at 37°C in 5% CO 2 ͞95% air for various times (0-96 h).…”

“…Within 24 h of the exposure in vitro, the pituitary tumor extract (PTE) led to aggregation of breast cancer cells, polarization of organelles, the formation of cell junctions and basement membrane, synthesis of mRNA for casein and lactalbumin, and overexpression of E-cadherin (6). The PTE was also effective on prostate cancer cells, inducing cellular changes and overexpression of E-cadherin and prostate-specific antigen.…”

Structural investigation of tumor differentiation factor

“…TDF induces the differentiation of human breast and prostate cancer cells in vitro and in vivo [1], [2]. However, TDF does not have any morphological or biochemical differentiation effect on fibroblasts or on kidney, hepatoma, and leukemic lymphocytic cell lines.…”