Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas.

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53(-/-) carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53(-/-) carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial-mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.

Behavioural and endocrinological changes in Syrian hamsters (Mesocricetus auratus) under domestication.

This study aimed to quantify differences in response to stress between two strains of Syrian hamsters to evaluate the consequences of domestication in this species by measuring behavioural traits in the open-field, adrenal gland masses (ADR), and faecal and blood corticosterone concentrations (CC). We studied a laboratory (lab)- and a wild-derived population (wild). The lab hamsters were significant heavier than the wild hamsters. The lab males had the highest ADR, and it was independent of their high body mass (BM). The ADR of lab females and wild hamsters was linearly dependent of BM. The lab males had the highest faecal and blood CC, whereas the lab females had the lowest CC. In the open field, the lab hamsters began later to groom, groomed shorter, groomed less frequently, began later to rear, reared longer and reared less frequently. In the lab population, females reared more often and groomed longer than males. The sex differences in the behaviours of the lab population and the differences between the populations mirror the differences neither in the ADR nor in the CC. The founder effect and the breeding history of lab Syrian hamsters are discussed as causes of the differences between the studied populations.

Janus kinase 2 is required for the initiation but not maintenance of prolactin-induced mammary cancer.

The prolactin receptor (PRLR), its associated Janus kinase 2 (Jak2) and the signal transducer and activator of transcription 5 (Stat5) are essential for normal mammary gland development. Owing to the upregulation of the PRLR and the local synthesis of its ligand in neoplastic cells, it has been proposed that PRL can act as a local growth factor in human breast cancers. This notion is supported by experimental evidence in transgenic mice, which showed that the mammary-specific expression of PRL contributes to carcinogenesis in vivo. To assess the importance of Jak2/Stat5 signaling during mammary cancer initiation and progression, we generated a PRL-induced mammary cancer model that allows the functional ablation of the Jak2 gene in the mammary epithelium before and after neoplastic transformation. Collectively, the results of this study show that the functional ablation of Jak2 protects against the onset of PRL-induced mammary tumorigenesis, suggesting that targeting this kinase is a relevant strategy for mammary cancer prevention. Surprisingly, Jak2 deficiency did not affect the growth and survival of PRL-induced mammary cancer cells in culture and in vivo. Consequently, Jak2 cannot be a sole therapeutic target to treat the established disease. PRL-induced mammary cancers exhibited an upregulation of ErbB2 and other ErbB receptor tyrosine kinases that may supersede the functionality of PRLR signaling through Jak2.

Stat5 activation inhibits prolactin-induced AP-1 activity: distinct prolactin-initiated signals in tumorigenesis dependent on cell context.

The essential role of prolactin (PRL) in normal mammary gland growth and differentiation has implicated this hormone in the development and progression of breast cancer. Although Stat5 is the best-characterized mediator of PRL signals, PRL also activates multiple other signals, whose roles in normal and pathologic processes are not well understood. We have shown that PRL stimulates activating protein-1 (AP-1) activity in breast cancer cells, and can cooperate with estradiol in this pathway. AP-1 modulates many processes critical for carcinogenesis, including cell proliferation, survival, transformation, invasion and angiogenesis, and is elevated in many neoplasms, including breast tumors. Here, we investigated the relationship between PRL signals to AP-1 and Stat5. We found that PRL activation of Stat5a and Stat5b, but not Stat1 or Stat3, reduced PRL signals to AP-1, without altering estradiol-induced AP-1 activity. The truncation mutant, Stat5/Delta53C, but not Stat5Y699F, was an effective inhibitor, consistent with a requirement for Stat5 dimerization and nuclear accumulation, but not its C-terminal transactivation activity. The association of Stat5 with AP-1 proteins suggests that this underlies the inhibition. Predictably, the ability of PRL to activate Stat5 and AP-1 was inversely related in mammary cell lines. Further, reduction of Stat5 protein with siRNA in T47D cells, which contain elevated Stat5, increased PRL-induced AP-1 signals, transcripts for the AP-1 target, matrix metalloproteinase-2 and associated invasive behavior. This study points to the importance of cell context in determining the spectrum of PRL-induced actions, which is critical for understanding the contributions of PRL to breast cancer.

Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Joint toxicity of triazine herbicides and organophosphate insecticides to the midge Chironomus tentans.

A series of recent studies demonstrated that the triazine herbicide atrazine, although not itself acutely toxic, potentiated the toxicity of certain organophosphate insecticides (OPs) to the midge Chironomus tentans. In the current study, a series of triazine herbicides and triazine herbicide degradation products were tested to determine if other triazines potentiate OP toxicity to midges. Chlorpyrifos and diazinon were the OPs tested. Toxicity tests were conducted using a factorial design and analysis of variance to statistically determine if each triazine had an effect on expected toxicity. Log-probit procedures were also used to evaluate the magnitude of change in median effective concentration (EC50) values during coexposure with each triazine. All of the triazine herbicides tested (atrazine, simazine, cyanazine, and hexazinone) were capable of potentiating the toxicity of the OPs, whereas the degradation products (s-triazine, deethylatrazine, and deisopropylatrazine) had less effect. In most cases, a triazine concentration of 100 microg/L was necessary to significantly increase OP toxicity, and higher concentrations of triazine caused a greater degree of potentiation. Changes in EC50 values ranged from no change to a 2.5-fold increase in toxicity. Generally, EC50 values changed by less than a factor of 2, indicating that the effect may be of limited concern in regard to future risk assessments of OPs.

Temporospatial expression of placental lactogen and prolactin-related protein-1 genes in the bovine placenta and uterus during pregnancy.

The anatomical location of binucleate cells (BNC) influences protein expression but not steroid synthesis in ruminants. In order to determine if BNC in disparate locations differentially express bovine placental lactogen (bPL) and prolactin-related protein-1 (bPRP-1), we quantitated bPL and bPRP-1 transcripts in placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues throughout pregnancy in the bovine using real-time reverse transcription PCR (RT-PCR) and in situ hybridization. Levels of both bPL and bPRP-1 transcripts at peri-implantation were significantly higher (P < 0.01) in the fetal membrane than in caruncular and intercaruncular tissues. Thereafter, mRNA for these related proteins demonstrated different spatial as well as temporal patterns of expression. Levels of bPRP-1 transcripts peaked at day 60 of pregnancy. Between day 60 and 100, bPRP-1 transcripts fell by approximately sevenfold (P < 0.01) in cotyledonary and intercotyledonary tissues, and fourfold in caruncular (P < 0.01) tissue. Levels of bPRP-1 transcripts remained low in the cotyledonary, intercotyledonary, and caruncular tissues until peripartum. In contrast, bPL expression in placentomes increased with progression of gestation (P < 0.01), but decreased in interplacentomal tissue around peripartum. To conclude, disparate patterns of bPRP-1 and bPL genes are transcribed in the placentomal and interplacentomal tissues during gestation in the bovine, suggesting that these prolactin-like hormones serve distinct functions and are regulated differently in the uteroplacental unit in this species.

Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics.

Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.

Bluetongue surveillance methods in the United States of America.

Historical surveillance for bluetongue virus (BTV) exposure in the United States of America (USA) has relied on periodical serological surveillance using samples collected from cattle at slaughter. Most of this surveillance has been focused on the north-eastern portion of the USA due to the lack of competent vectors of BTV in this region. For most of the states tested in this region, the prevalence of seropositive animals has been less than 2%. Recently, a study was conducted in north-central USA using sentinel cattle herds. Results of serological testing showed an increasing gradient of exposure from north to south. In addition, detection of Culicoides sonorensis showed a similar gradient with detection in the northern areas being relatively rare. The results of these studies indicate that cattle herds in the northern and north-eastern areas of the USA are likely to be free of BTV.

Expression of prolactin-related protein I at the fetomaternal interface during the implantation period in cows.

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.

Monthly model for genetic evaluation of laying hens. II. Random regression.

1. We investigated the use of monthly production records for genetic evaluation of laying hens, derived from a test day model with random regression in dairy cattle and compared it with other models. 2. Records of 6450 hens, daughters of 180 sires and 1335 dams, were analysed using a model with restricted maximum likelihood (REML): traits considered were monthly and cumulative egg production. Five models were studied: (1) random regression with covariates derived from the regression of Ali and Schaeffer (Canadian Journal of Animal Science, 67: 637-644, 1987) (RRMAS), (2) random regression with covariates derived from quartic polynomial (RRMP4), (3) fixed regression with covariates derived from Ali and Schaeffer (FRM), (4) multiple trait (MTM) and (5) cumulative (CM). 3. The models were compared on the basis of Spearman rank correlations of individual breeding values and sire breeding values estimated from subsets of full-sib split data. The hens (about 10% per generation) which ranked highest on their estimated breeding values from different models were compared phenotypically with their full records. 4. The estimates of heritability resulting from RRMP4 were biased upward from the estimates obtained from MTM, so this model was discarded. The heritabilities for monthly productions from RRMAS and MTM showed a similar pattern. They were high for the 1st month of production, decreased to their lowest value at about month 5 of production and increased again to the end of lay. 5. Spearman rank correlations between animal breeding values estimated by monthly models (RRMAS, FRM and MTM) were high, between 0.91 and 0.98, whereas those between estimates of monthly models and CM were lower, from 0.85 to 0.87. The correlations estimated either from intermittent months of measurements (odd vs even months) or full records were generally high, from 0.93 to 0.99. Information from odd months of production could be sufficient for cost-efficient recording schemes. The RRMAS generally had the highest correlation of sire breeding values between subsets of full-sib records, followed by MTM, RM and CM. Monthly models selected hens with higher productivity than the cumulative model. 6. In conclusion, genetic evaluation based on monthly production may be better than using cumulative production and RRMAS appeared to be the best among the models tested here.

Bioavailability of sediment-associated benzo(a)pyrene within single- versus multiple-species systems.

A series of experiments were conducted with benzo(a)pyrene (B(a)P) spiked sediments to determine if bioavailability of sediment-associated contaminants is affected by multiple species interactions. Three benthic invertebrates, Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus, were exposed to sediments spiked with radiolabeled B(a)P that was aged for 60 days. Organisms were introduced into the spiked sediments in single, binary, and ternary combinations. Changes in bioavailability were then determined for each species by estimating uptake clearance coefficients (ks) and bioaccumulation factors (BAFs) during 7-day exposures. In general, there was a trend toward lower ks values in binary and ternary exposures compared to the single-species systems. In contrast, BAF estimates were more variable with fewer significant differences noted among treatments. BAF estimates were highest for L. variegatus followed by C. tentans and H. azetca and appear to be dependent on specific feeding and habitat requirements as well as the relative biotransformation/elimination potential of each species. Overall, these results suggest that animal-animal interactions may be important to consider when estimating bioavailability of sediment-bound chemicals.

Pregnancy outcome following gestational exposure to venlafaxine: a multicenter prospective controlled study.

OBJECTIVE: Because there are no studies available on the safety of venlafaxine during pregnancy, the authors' goal in this study was to determine whether venlafaxine increases the risk for major malformations. METHOD: Data on 150 women exposed to venlafaxine during pregnancy in seven pregnancy counseling centers were compared with data from studies of pregnant women who 1) received selective serotonin reuptake inhibitor antidepressants (SSRIs) (N=150) and 2) who received nonteratogenic drugs (N=150). RESULTS: Among the 150 women who were exposed to venlafaxine during pregnancy, 125 had live births, 18 had spontaneous abortions, and seven had therapeutic abortions; two of the babies had major malformations. There were no significant differences between these women and the two comparison groups on any of the measures analyzed. CONCLUSIONS: These results suggest that the use of venlafaxine during pregnancy does not increase the rates of major malformations above the baseline rate of 1%-3%.

Molecular dynamics simulation of n-dodecyl phosphate aggregate structures.

Aggregates of n-dodecyl phosphate present an attractive model system of simple phospholipid amphiphile supramolecular structures for study by molecular dynamics simulation, since these systems have previously been studied experimentally under various conditions. A detailed molecular dynamics description of the properties of planar bilayer membranes (as a model for unilamellar vesicular membranes) and spherical micelles under various simulated conditions is presented. It is shown that the united-atom model of GROMOS96 applying the force-field parameter set 43A2 for biomolecular systems yields properties in agreement with experimental ones in most cases. Hydrogen bonding plays a role in stabilizing the bilayer aggregates at low pH, but not for the micelles, which are energetically favoured at high pH. NMR -S(CD) order parameters for a lipid bilayer system, the diffusion of amphiphiles within aggregates and of counterions, and lifetimes of hydrogen bonds between amphiphiles and to water are estimated from the MD simulations.

Chemical and biological availability of sediment-sorbed benzo[a]pyrene and hexachlorobiphenyl.

This study examined the chemical and biological availability of two nonpolar organic compounds, benzo[a]pyrene (BaP) and hexachlorobiphenyl (HCBP), from a spiked sediment that was aged for varying amounts of time. Chemical availability was evaluated using four different solvent combinations to extract chemicals from the sediment. The extractability of BaP and HCBP from sediment using traditional solvents was then compared to the transfer efficiency (TE) of a benthic invertebrate (Lumbriculus variegatus) to relate chemical extractability to bioavailability in the organisms. Results indicated that water was the solvent that best approximated bioavailability for BaP, whereas comparisons for HCBP were inappropriate, because TE values exceeded 100%. The inability to obtain a reasonable TE estimate for HCBP was most likely due to the fact that the oligochaetes received a major portion of their uptake from interstitial water instead of ingestion of sediment particles, which invalidated an important assumption of the TE model. Overall, the results of this study indicate that exhaustive chemical extractions may be an inaccurate representation of the bioavailable fractions for some contaminants.

Monthly model for genetic evaluation of laying hens 1. Fixed regression.

1. This paper addresses the possibility of using a monthly model for the genetic evaluation of laying hens, based on the definition of a test day model with fixed regression as used in dairy cattle, in which monthly records were treated as repeated measurements of the same trait. 2. Production records of 6450 hens, daughters of 180 sires and 1335 dams were analysed using an animal model with restricted maximum likelihood (REML). The traits considered were individual monthly egg production and cumulative egg production in 11 months. Four different models were fitted to various combinations of monthly and cumulative records. The covariates were derived from the regression of Ali and Schaeffer (1987). 3. Spearman rank correlations were computed to compare breeding values from different models. Two types of correlations were computed: between individual breeding values and between sire breeding values based on subsets of full-sib records. 4. The results indicated that a monthly model with nested covariates produced higher heritability and permanent environmental variance than the models with non-nested or without covariates. The estimates of heritability obtained from monthly model were lower than the estimates from the cumulative model. The monthly model resulted in higher correlations of sire breeding values between two subsets of full-sib records than those from cumulative models. 5. In conclusion, the monthly model with nested covariates appears to be better than the model with non-nested covariates or without covariate. Although the heritability estimates obtained from the monthly model were lower, the monthly model with nested covariates could be better than the cumulative model for genetic evaluation of laying hens in the 1st cycle of laying period when using either full or part records. The use of information from odd months of production could be of interest for the evaluation of full records.

Taking drugs during pregnancy. How safe are the unsafe?

QUESTION: I prescribed misoprostol to one of my patients with a peptic ulcer. When she found out she was pregnant while on the drug, both she and, admittedly, I were very scared to learn that the drug is teratogenic in that it causes Möbius syndrome. How great is the risk? ANSWER: Very small. Although women who use misoprostol during the first trimester have a 30-fold higher risk of having babies with Möbius syndrome, the malformation is so rare that, even if you see 1000 women who took misoprostol during embryogenesis, you might not see a single child with the syndrome. It is crucial to explain the size of the risk; otherwise women tend to believe the risk is huge even when, in fact, it is hardly measurable.

Prolactin receptor heterogeneity: processing and signalling of the long and short isoforms during development.

During development, the fetus is exposed to prolactin activity from the placenta, as well as from the developing fetal pituitary. Distinct prolactin receptor isoforms, having different cytoplasmic domains generated by alternative splicing, are expressed as development proceeds at different levels in different organs. The "long" receptors are able to mediate transduction of all signals examined, in contrast with the "short" isoforms, whose truncated cytoplasmic domains are able to mediate a much smaller repertoire of signals and can act as dominant negatives. Our studies demonstrate that, although these forms share internalization mechanisms, the long form is internalized faster, resulting in more rapid down-regulation of this form. In order to examine the mechanisms by which prolactin may exert trophic effects on its target tissues during development, we have examined the signalling pathways through which prolactin binding to the long receptor regulates the transcription of cyclin D1. Our studies reveal the importance of the JAK/STAT (Janus kinase/signal transduction and activators of transcription) pathway, and the complexity of prolactin signalling to this promoter.

Dysregulation of mammary Stats 1,3 and 5 and PRL receptors by overexpression of TGFalpha.

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.