A cDNA clone of 1.1 kb encoding a 108-aa polypeptide was isolated from a human pituitary cDNA library by expression cloning. This protein was named tumor differentiation factor (TDF). The recombinant TDF protein and a 20-aa peptide, P1, selected from the ORF of the gene, induced morphological and biochemical changes consistent with differentiation of human breast and prostate cancer cells. Fibroblast, kidney, hepatoma, and leukemic lymphocytic cell lines were unaffected. Breast and prostate cancer cells aggregated in spheroidlike structures within 24 h of exposure to TDF. This effect was abrogated by a specific affinity-purified rabbit polyclonal anti-P1 Ab. E-cadherin expression was increased in a dose-dependent manner by TDF. Treatment of MCF7 cells with TDF led to production of a lactalbumin-related protein. Peptide P1 significantly decreased the growth of androgen-independent DU145 prostate cancer in severe combined immunodeficient mice. The presence of TDF protein in human sera was detected by the anti-P1 Ab, suggesting a role of TDF in endocrine metabolism. The fact that all activities of TDF can be mimicked by a peptide derived from the encoding TDF sequence opens the possibility of therapeutic applications.M alignant transformation is characterized by the uncoupling of proliferation and differentiation leading to continuing multiplication of cells and the impairment of their ability to progress to complete normal differentiation. Restoration of differentiation of malignant cells has long been considered a potential therapy of cancer (1-5).We have previously reported that an alkaline extract of rat mammosomatotropic tumor MtTW10 induced morphological and biochemical changes in rat and human breast cancer cells indicative of differentiation. 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.Because this activity was not reproduced by any of the known pituitary hormones or other growth factors, studied alone or in combination, we assumed that the PTE contained a factor, which we called tumor differentiation factor (TDF). Our attempts to isolate and purify this factor by using conventional chromatographic procedures were unsuccessful. Now, using expression cloning, we report the isolation of a human cDNA clone encoding a protein responsible for this tumordifferentiating activity. We have sequenced the clone. Computer analysis indicates that it encodes a polypeptide with no significant homology to any other known sequence in the GenBank database. Our results support the proposition that TDF is a previously unrecognized pituitary factor. Materials and MethodsPituitary cDNA Library. A cDNA library prepared from a growth hormone-producing human ...
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.
The data presented here provide the first evidence for PAR gene cellular function and its possible implication in malignant transformation.
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