Ameae M. Walker

Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids

Soluble formazan assays are widely used for cell number assessment. However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading. In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids. To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Identification of the major site of rat prolactin phosphorylation as serine 177.

Phosphorylation of prolactin by endogenous protein kinases within isolated secretory granules was shown to result in the production of both phosphoserine and phosphothreonine residues. The majority of the radiolabel was determined to be present in the C terminus of the molecule after specific cleavage with glandular kallikrein. Glandular kallikrein cleaves in three places at the C terminus, liberating three small peptides, only one of which contains a phosphorylatable residue. Sequencing of this phosphopeptide showed it to be Arg-Lys. Thus the major site of prolactin phosphorylation was determined to be serine 177. Using a synthetic peptide equivalent to this region of the molecule (Ser-Val), serine 177 was demonstrated to be a substrate for protein kinase A as well as for one of the endogenous granule kinases. Inclusion of the synthetic peptide in an endogenous granule phosphorylation reaction resulted in competition for the kinase and reduced phosphorylation of prolactin. Protein kinase A phosphorylation of purified prolactin resulted in the production of only phosphoserine and primarily the most abundant (monophosphorylated) variant. We conclude that serine 177 is the major in vivo phosphorylation site of rat prolactin and that phosphorylation of this site can be reproduced by protein kinase A in vitro. The minor threonine phosphorylation site was demonstrated by two-dimensional tryptic peptide mapping and mass analysis to be either threonine 58 or 63, both of which are contained within a single peptide.

Phosphorylated and nonphosphorylated prolactin isoforms

Specific posttranslational modifications o f monomer PRL produce a number of hormone variants. This review describes the current state of knowledge for the nonphosphorylated and phosphorylated 24-kD monomer forms. Of particular interest and significance for our future understanding of PRL endocrinology are the studies showing their differential release in response to different physiologic signals and their different activities in target tissues. In at least two target tissues, the monophosphorylated variant seems to act as an antagonist to the unmodified hormone.

Trans-epithelial immune cell transfer during suckling modulates delayed-type hypersensitivity in recipients as a function of gender.

Introduction Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult. Methods Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls. Results Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3–4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3–4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males. Discussion These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted.

Dissociation of Janus Kinase 2 and Signal Transducer and Activator of Transcription 5 Activation after Treatment of Nb2 Cells with a Molecular Mimic of Phosphorylated Prolactin1

We have previously demonstrated that phosphorylated PRL acts as an antagonist to the Nb2 proliferative activities of unmodified PRL. A molecular mimic of phosphorylated PRL, which substitutes an aspartate residue for the normally phosphorylated serine (serine 179), has the same properties. Because it takes less than one fourth the amount of phosphorylated hormone, or the aspartate mutant, to block the proliferative activity of unmodified hormone, we have investigated whether the high potency of the aspartate mutant is achieved by the production of an alternate and interfering intracellular signal cascade. Nb2 cells were exposed to 5 or 500 ng/ml human NIDDK PRL, wild-type recombinant PRL (unmodified PRL), or aspartate mutant PRL (pseudophosphorylated PRL) for 1, 5, or 10 min at 37 C. At 5 ng/ml and 10 min, wild-type recombinant PRL showed greater activation of Janus kinase 2 (JAK 2) than the NIDDK preparation. This is consistent with a previous report of higher proliferative activity for the wild-type hor...

p21-activated protein kinase γ-PAK in pituitary secretory granules phosphorylates prolactin

p21‐activated protein kinase γ‐PAK phosphorylates prolactin (PRL) in rat pituitary secretory granules on Ser‐177 and on the equivalent site, Ser‐179, in recombinant human PRL. This is shown by comparison of phosphopeptide maps with the human PRL mutant S179D. γ‐PAK is present in rat and bovine granules as identified by in‐gel phosphorylation of histone H4, and by immunoblotting. Thus, phosphorylation of PRL by γ‐PAK in granules produces the PRL molecule that has been shown to antagonize the growth‐promoting activity of unmodified PRL, and is consistent with the identified role of γ‐PAK in the induction and maintenance of cytostasis.

Short form 1b human prolactin receptor down-regulates expression of the long form

Alternative splicing produces different human prolactin (PRL) receptors. These include a long form (LF) and two short forms (SF1a and SF1b). The SFs of the receptor can act as dominant negatives for PRL effector function through the LF. This is proposed to be due to LF-SF heterodimerization and resultant interference with LF-LF dimer signaling. We, along with others, have provided evidence for LF-SF heterodimerization of the human receptors in support of this mechanism, along with others. However, to further investigate the ways SF may influence LF function, we co-transfected human embryonic kidney 293 cells with vectors coding for tagged (green fluorescent protein (GFP) or luciferase) LF alone or plus untagged SF1b and measured LF-GFP intensity, LF-luciferase activity, and LF mRNA 48 h later. Equal amounts of SF1b cDNA decreased LF-GFP fluorescence intensity, LF-luciferase activity, and LF mRNA by 80-100%. Similar co-transfections with untagged LF had no significant effect on tagged LF expression. Use of hygromycin showed degradation of already formed protein was the same for LF-luciferase alone and LF-luciferase with SF1b. Inhibition of mRNA synthesis, on the other hand, showed that SF1b expression accelerated LF mRNA degradation two- to three-fold. SF1b also down-regulated expression of endogenous LF mRNA in T47D breast cancer cells and opposed an increase in cell number resulting from transfection with extra LF alone. These results demonstrate a previously unrecognized mechanism whereby SF1b affects the end result of signaling through the LF receptor. The effects on cell number also support the concept that the LF:SF1b ratio may be relevant to tumor growth.

Structure-Function Relationships in Prolactin

In this chapter, it is the authors’ intent to provide an update on structure-function relationships for PRL. We have only covered those PRL varieties for which additional information has been gained since the excellent and comprehensive review by Sinha in 1995 (1). Focus is on forms for which a different biological activity is becoming evident, although attention is also drawn to reports of some new PRLs, the existence of which may help to explain old controversies. Also included, are mutational studies which have contributed greatly to our understanding of hormone-receptor interactions and, therefore, to the potential impact of specific posttranslational modifications on biological activity. We have attempted to meld old and new data on each form of PRL into a comprehensive view whenever we felt that was possible. When such was not possible, we have suggested lines of investigation which may resolve current controversies.

S179D prolactin : Antagonistic agony!

The aims of this review are three-fold: first, to collate what is known about the production and activities of phosphorylated prolactin (PRL), the latter largely, but not exclusively, as illustrated through the use of the molecular mimic, S179D PRL; second, to apply this and related knowledge to produce an updated model of prolactin-receptor interactions that may apply to other members of this cytokine super-family; and third, to promote a shift in the current paradigm for the development of clinically important growth antagonists. This third aim explains the title since, based on results with S179D PRL, it is proposed that agents which signal to antagonistic ends may be better therapeutics than pure antagonists-hence antagonistic agony. Since S179D PRL is not a pure antagonist, we have proposed the term selective prolactin receptor modulator (SPeRM) for this and like molecules.