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Yale University School of Medicine, Department of Pharmacology, New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Dr. Priscilla S. Dannies, Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street, New Haven, Connecticut 06520.
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Identification of this activity will require purification of the factor(s). The original isolations of biologically active peptides from the hypothalamus were heroic tasks that involved collecting hundreds of thousands of bovine hypothalami (8, 9, 10). In this issue, Ben-Jonathan and her co-workers (11) have used a different approach to obtain material to use to isolate the PRL-releasing activity by taking advantage of advances in biotechnology that have occurred since the first factor was isolated.
It is now possible to create designer cell lines that express some of the differentiated properties of the tissues of origin. Cell lines with differentiated properties were developed from tumors in the 1960s (12), but a limiting step has been obtaining tumors of the desired cells in order to create the lines. Now, however, transgenic mice can be created with tumors in specific cell types. The tumors grow because a viral protein that is tumorigenic for most cells, such as the SV40 large T antigen, is expressed in the cells, and tissue-specific promoters ensure that the mice express this tumor-causing protein only in desired cells. POMC is expressed only in certain cells of the pituitary gland and the hypothalamus, so when the POMC promoter is attached to the SV40 T antigen and incorporated into a transgenic mouse genome, cell-specific transcription factors that cause production of POMC from the endogenous POMC promoter will also cause production of SV40 T antigen from the exogenous POMC promoter.
The intermediate lobe of the pituitary gland of the rat consists of primarily POMC-producing cells, so it was a reasonable assumption that the PRL-releasing activity might also be produced by these cells, in which case tumors caused by expression of a protein driven by a POMC promoter might be a good choice for a source of cells that also made the releasing activity. The fragment of POMC promoter used to induce to tumors worked best in the intermediate lobe, rather than the anterior pituitary gland or the hypothalamus, which was just what was needed for this project. The mice grew large intermediate lobe tumors. Ben-Jonathan and co-workers found that extracts of the tumors had PRL-releasing activity and then set about deriving stable cell lines as more accessible sources of the activity. They isolated a cell line that expressed POMC and T antigen as predicted, but that had little or no PRL-releasing activity; the cells might have lost the ability to make the factor. They did obtain a cell line that produces PRL-releasing activity, but unexpectedly, it expresses neither T antigen nor POMC. It isn’t clear why this cell line grows continually in culture if not expressing T antigen; a fortuitous spontaneous mutation may have occurred. Because no other differentiated functions have been identified except the PRL-releasing activity, the lineage of these cells is not yet known. So the rational use of biotechnology led to a promising cell line for a source of PRL-releasing activity, but not for the expected reasons, and luck seems to have played a part.
What remains to be done to demonstrate that the activity is a releasing factor? The authors now refer to it as a regulatory factor. In addition to purification and characterization of the activity, there are two important questions. One is relatively easy: does this factor stimulate rapid release of PRL? In the paper in this issue, the authors measured stimulation of PRL secretion into the medium over an 18-h period from GH3 cells and normal lactotrophs. GH3 cells ordinarily contain amounts of PRL equivalent to 1–2 h worth of secretion, so marked increases in PRL secretion over 18 h reflect increases in PRL synthesis. The authors also assayed PRL gene activity by measuring luciferase activity under the control of the PRL promoter. In most cases in GH3 cells, increases in PRL synthesis reflect increases in PRL mRNA transcription, so it is not surprising that stimulation of secretion over 18 h is similar to stimulation of luciferase activity. A long-term effect on synthesis, however, need not indicate a short-term effect on release. Compounds, for example, steroids, may affect PRL synthesis without rapidly affecting PRL release, so it is important to know if the activity produced by the new cell line stimulates PRL release as rapidly as suckling increases PRL release in animals.
The second question is whether the cell line is expressing the same PRL-releasing activity that the normal intermediate lobe of the pituitary gland does. Many factors stimulate PRL production. Changes in protein expression that allowed this cell line to grow may have caused expression of a factor not otherwise produced by the intermediate lobe. If the fortune of these workers continues, these cells are producing the same factor that the intermediate lobe produces, and a new releasing factor will be characterized.
Received October 8, 1997.
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  M. Maruyama, H. Matsumoto, K. Fujiwara, C. Kitada, S. Hinuma, H. Onda, M. Fujino, and K. Inoue Immunocytochemical Localization of Prolactin-Releasing Peptide in the Rat Brain Endocrinology, May 1, 1999; 140(5): 2326 - 2333. [Abstract] [Full Text]
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