Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis.

Regions where one type of epithelium replaces another (metaplasia) have a predilection for cancer formation. Environmental factors are closely linked to metaplastic carcinogenesis. In particular, cervical cancers associated with human papillomavirus (HPV) infection develop primarily at the transformation zone, a region where metaplastic squamous cells are detected in otherwise columnar epithelial-lined endocervical glands. Previously, we reported estrogen-induced multistage vaginal and cervical carcinogenesis in transgenic mice expressing HPV16 oncogenes in basal squamous epithelial cells. In the present study to investigate the threshold neoplastic response to exogenous estrogen, we treated groups of transgenic mice with lower hormone doses. A 5-fold reduction in estrogen dose induced squamous carcinogenesis solely at the cervical transformation zone compared with other reproductive tract sites. Further study delineated stages of transformation zone carcinogenesis, including formation of hyperplastic lower uterine glands and emergence of multiple foci of squamous metaplasia from individual stem-like glandular reserve cells, followed by neoplastic progression of metaplasia to dysplasia and squamous cancer. We propose that a combination of low-dose estrogen and low-level HPV oncogene expression biases transformation zone glandular reserve cells toward squamous rather than columnar epithelial fate decisions. Synergistic activation of proliferation by viral oncoprotein cell cycle dysregulation and estrogen receptor signaling, together with altered paracrine stromal-epithelial interactions, may conspire to support and promote neoplastic progression and cancer formation.

Neonatal administration of prolactin antiserum alters the developmental pattern of T- and B-lymphocytes in the thymus and spleen of BALB/c female mice.

We have evaluated the effect of neonatal administration of mouse prolactin (PRL) antiserum on the developmental expression of T- and B-lymphocytes in the thymus and spleen of female BALB/c mice. Newborn female mice were injected subcutaneously with a 50-microliters aliquot of PRL antiserum or normal rabbit serum on days 1, 2, and 3. On neonatal day 5, the PRL antiserum-treated group had a significantly (P less than 0.05) increased population of cells in the thymus and the spleen that were positive for Thy-1.2 and for L3T4. Increases in Thy-1.2- and L3T4-positive cells in the thymus were detectable also on days 8 and 14 in mice that received the PRL antiserum and in mice injected with bromocriptine, a dopamine agonist that inhibits PRL release from the anterior pituitary. On neonatal days 21, 28, and 32, there were no significant differences in the percentage of cells positive for Thy-1.2, Ly-2 (formerly Lyt-2), or L3T4 antigens in the thymus. However, there were significant increases in the percentage of Thy-1.2- and L3T4-positive spleen cells in the bromocriptine-treated group at all times monitored and in the PRL antiserum-treated group except on day 14. In addition, the percentage of splenocytes that were positive for IgG was significantly increased in the PRL antiserum-treatment group on days 8-28, although not on neonatal day 32. Of tissues known to contain PRL receptors, neonatal administration of PRL antiserum or bromocriptine resulted in a significant alteration in the wet weight of spleen and liver, with no significant effect in thymus, heart, and kidney. Pituitary implants also resulted in a significant increase in both concanavalin A- and lipopolysaccharide-stimulated thymidine incorporation into murine splenic lymphocytes prepared from 45-day-old female mice. These data extend the role of PRL as an immunomodulator of adult lymphocyte function to a role in the developmental expression of T- and B-lymphocyte populations in the thymus and spleen of mice.

Studies on dissociation of mouse prolactin from mouse hepatic receptors.

The influence of pH, temperature, ethylene glycol, urea, chaotropic anions and excess unlabelled secreted mouse prolactin (smPRL) on the dissociation kinetics of 125I-iodosmPRL from mouse hepatic receptors was investigated. The destabilization of smPRL-receptor complexes by chaotropic anions followed the typical trend of the Hofmeister series: I- greater than Br- greater than Cl- greater than F-. Increasing the temperature of the dissociation reaction from 8 degrees C to 23 degrees C and 30 degrees C caused partial dissociation of 125I-iodosmPRL-receptor complexes. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was pH dependent, with the slowest rate of dissociation occurring at pH 8 and the fastest rate of dissociation occurring at pH 5 and 6. Both ethylene glycol and urea accelerated the rate of dissociation of 125I-iodosmPRL from mouse hepatic receptors in a concentration-dependent manner. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was 6-fold faster in the presence of excess unlabelled smPRL than in its absence. The results of these investigations suggest that both protonation/de-protonation reactions and hydrophobic interactions play important roles in stabilizing the smPRL-receptor complex. In addition, they suggest that cooperative interactions may be involved in the binding of smPRL to mouse hepatic receptors.

Thermodynamics and kinetics of mouse prolactin-hepatic receptor interaction.

Kinetic and thermodynamic parameters associated with the binding of secreted mouse prolactin (smPRL) to mouse hepatic receptors were investigated. When the reaction temperature was increased from 8 degrees C to 37 degrees C, the association rate constant k+1, increased approximately 5-fold, from 2.3 X 10(4) M-1 . S-1 to 12.6 X 10(4) M-1 . S-1. An Arrhenius plot indicated that there was a linear relationship between ln (k+1) and 1/T. When the reaction temperature was increased from 8 degrees C to 37 degrees C, the equilibrium binding constant, Ka, decreased approximately 1.5-fold, from 2.8 X 10(8) M-1 to 1.9 X 10(8) M-1. When the pH of the binding reaction was lowered from 9.0 to 6.2, Ka decreased approximately 3-fold, from 2.6 X 10(8) M-1 to 0.9 X 10(8) M-1. Transition state thermodynamic parameters for the formation of the smPRL-receptor complex, represented by delta G+', delta H+' and delta S+', were +45.7 kJ/mol, +41.2 kJ/mol and -15.1 J/(mol . K), respectively. Parameters for the equilibrium reaction, described by delta G0', delta H0' and delta S0', were -47.6 kJ/mol, -10.6 kJ/mol and +124 J/(mol . K), respectively. Over the temperature range studied, a Van't Hoff plot of the binding constants demonstrated a linear relationship between ln (ka) and 1/T, indicating that changes in enthalpy for the binding reaction were temperature independent. The binding reaction was largely entropically driven (delta S0' greater than 0), suggesting that hydrophobic interactions are involved in forming the smPRL-receptor complex.

Interaction of mouse prolactin with mouse hepatic receptors.

Lactogenic receptors are usually studied in heterologous systems where prolactin is derived from one species and receptors prepared from another. In such systems the foreign prolactin could be seen as a growth hormone by the host tissue. We have therefore developed a homologous radioreceptor assay using secreted mouse prolactin (smPRL) and mouse hepatic receptors. In this system, monovalent anions augment the smPRL-receptor interaction in the order F- greater than Cl- greater than Br- greater than I-. Divalent cations (Mg2+, Ca2+, Sr2+), phosphate and acetate also increase smPRL binding. Temperature and pH optima are at 8 degrees C and pH 8.3, respectively. Under optimum conditions, the percent total, specific and nonspecific binding are 55%, 45% and 10%, respectively. At infinite receptor concentration the maximum specific bindability of labeled smPRL is 50%. The effects of ions on binding of smPRL to the receptor show that hydrophobic forces participate in smPRL-receptor coupling. The biphasic dissociation kinetics show initial and final rate constants of 1.56 X 10(-4)/s and 7.62 X 10(-6)/s, respectively. The lactogenic receptor does not bind mouse growth hormone; however, it binds both mouse placental lactogen (mPL) and smPRL with equilibrium association constants of 3.90 X 10(8) M-1 and 2.25 X 10(8) M-1, respectively, suggesting that smPRL and mPL share biological roles by acting through the same receptor.

Secreted mouse prolactin (PRL) and stored ovine PRL. II. Role of amides in receptor binding and immunoreactivity.

Native PRL and des-amido forms 1, 2, and 3 were tested for their individual immunochemical and receptor-binding abilities. The results show that deamidation of either secreted mouse PRL or stored ovine PRL alters their binding in a radioreceptor assay. For each accumulated deamidation there was a statistically significant (P less than 0.05) decrease in the binding potency of either stored ovine PRL or secreted mouse PRL to a cell membrane receptor preparation. RIAs indicated that there was a statistically significant (P less than 0.05) decrease in PRL's immunopotency toward polyclonal antisera only when select residues deamidated. This study suggests that all the asn/gln residues which deamidate in order to make des-amido forms 1, 2, and 3 constitute part of the receptor-binding domains of both secreted and stored PRLs, while only a fraction of those same residues form portions of PRL's antigenic sites. Thus PRL's receptor-binding surface is separated from its antigenic sites, with only partial overlap being indicated. Our data also indicate that there are no major structural differences in the receptor-binding domains of secreted and stored PRLs. We also see that the receptor-binding domain of PRL has been highly conserved throughout evolution. Since the binding affinity of PRL to a membrane-bound receptor can be altered through deamidation, we view the process as a plausible regulatory mechanism for controlling the quantitative action of PRL at a given target organ.

Secreted mouse prolactin (PRL) and stored ovine PRL. I. Biochemical characterization, isolation, and purification of their electrophoretic isoforms.

The biochemical nature of the electroisomers of secreted mouse PRL and stored ovine PRL were compared. When examined on alkaline polyacrylamide gels, they exhibited electrophoretic heterogeneity. The electrophoretic isomers had the same molecular size by examination of Ferguson plots and therefore differed only in net negative charge at alkaline pH. There was no apparent charge heterogeneity in the PRL preparations when they were electrophoresed at a pH below the pKa of the side-chain carboxyl groups of aspartic acid and glutamic acid; however, they exhibited size heterogeneity owing to aggregation. The slowest migrating electroisomers (from alkaline gels) converted spontaneously into faster migrating forms at 37 C in either acid (pH 4.0) or alkaline (pH 8.0) environments. The rate constant (determined at 37 C) for the transformation of the native PRL into faster migrating isoforms was 10.76 X 10(6) sec-1 at pH 8.0 and 0.50 X 10(6) sec-1 at pH 4.0. When secreted mouse PRL was incubated in alkaline (pH 10.0) conditions at 25 C, ammonia was released as the conversion reaction occurred. These results indicate that the electroisomers separated at alkaline pH are charge isoforms differing only in the number of free carboxyl groups of glutamic and/or aspartic acid residues. The des-amido isoforms were fractionated and purified. Polyacrylamide gel electrophoresis at alkaline pH was used to separate the charge isomers from each other. Each isoform was then excised from the separating matrix and recovered via electrophoretic elution. The homogeneity of each isoform was monitored by polyacrylamide gel electrophoresis.

Protein hormones: detection and extraction of functional forms from alkaline polyacrylamide gels.

A method is presented for rapidly staining zones (30 min) in alkaline polyacrylamide gels in the absence of fixatives. Native functional proteins are recovered in homogeneous form after excision of the visible zones from the polyacrylamide matrix. Removal of dye from excised zones is facilitated because only the surface of the gel is stained. Biological activity is then recovered from the gel slices by simple diffusion. The technique makes use of the sensitivity of Coomassie Brilliant Blue G-250 for the detection of proteins (less than 1 microgram). Structural variants of prolactin are isolated and recovered by this method. The method is applicable to studies requiring analytical and semi-preparative electrophoresis of proteins, especially pituitary hormones.

Homologous somatotropin radioreceptor assay utilizing recombinant bovine growth hormone.

A homologous radioreceptor assay using recombinant bovine growth hormone and bovine liver membranes is described. The total specific binding of 125I-labeled recombinant bovine growth hormone to the 100 000 X g pellet was 48% in 24 h at 25 degrees C. Hormone binding was partially reversible, with 40% of the radiolabeled hormone being irreversibly bound. The amount of specific binding varied with assay pH, with the optimum occurring at pH 7.8. Specific binding was temperature-dependent, with greater specific binding occurring at 25 degrees C than at 5 degrees C or 37 degrees C during a 24 h period. Recombinant bovine growth hormone, human growth hormone, ovine growth hormone and recombinant porcine growth hormone competed effectively with 125I-labeled recombinant bovine growth hormone for binding sites, while bovine prolactin and ovine prolactin were needed in amounts 10(6)-fold the concentration of recombinant bovine growth hormone to displace the radiolabeled hormone. Surprisingly, human placental lactogen did not displace the radiolabeled hormone.