Longitudinal association of prepubertal urinary phthalate metabolite concentrations with pubertal progression among a cohort of boys.

BACKGROUND: Epidemiological studies have reported associations of anti-androgenic phthalate metabolite concentrations with later onset of male puberty, but few have assessed associations with progression. OBJECTIVES: We examined the association of prepubertal urinary phthalate metabolite concentrations with trajectories of pubertal progression among Russian boys. METHODS: At enrollment (ages 8-9 years), medical history, dietary, and demographic information were collected. At entry and annually to age 19 years, physical examinations including testicular volume (TV) were performed and spot urines collected. Each boy's prepubertal urine samples were pooled, and 15 phthalate metabolites were quantified by isotope dilution LC-MS/MS at Moscow State University. Metabolites of anti-androgenic parent phthalates were included: butylbenzyl (BBzP), di-n-butyl (DnBP), diisobutyl (DiBP), di(2-ethylhexyl) (DEHP) and diisononyl (DiNP) phthalates. We calculated the molar sums of DEHP, DiNP, and all AAP metabolites. We used group-based trajectory models (GBTMs) to identify subgroups of boys who followed similar pubertal trajectories from ages 8-19 years based on annual TV. We used multinomial and ordinal regression models to evaluate whether prepubertal log-transformed phthalate metabolite concentrations were associated with slower or faster pubertal progression trajectories, adjusting for covariates. RESULTS: 304 boys contributed a total of 752 prepubertal urine samples (median 2, range: 1-6) for creation of individual pools. The median length of follow-up was 10.0 years; 79% of boys were followed beyond age 15. We identified three pubertal progression groups: slower (34%), moderate (43%), and faster (23%) progression. A standard deviation increase in urinary log-monobenzyl phthalate (MBzP) concentrations was associated with higher adjusted odds of being in the slow versus faster pubertal progression trajectory (aOR 1.47, 95% CI 1.06-2.04). None of the other phthalate metabolites were associated with pubertal progression. CONCLUSIONS: On average, boys with higher concentrations of prepubertal urinary MBzP had a slower tempo of pubertal progression, perhaps attributable to the disruption of androgen-dependent biological pathways.

The histopathology of a human mesenchymal stem cell experimental tumor model: support for an hMSC origin for Ewing's sarcoma?

Sarcomas display varied degrees of karyotypic abnormality, vascularity and mesenchymal differentiation. We have reported that a strain of telomerized adult human bone marrow mesenchymal stem cells (hMSC-TERT20) spontaneously evolved a tumorigenic phenotype after long-term continuous culture. We asked to what extent our hMSC-TERT20 derived tumors reflected events found in human sarcomas using routine histopathological procedures. Early versus late passage hMSC-TERT20 cultures persistently expressed mesenchymal lineage proteins e.g. CD105, CD44, CD99 and vimentin. However, late passage cultures, showed increased immunohistochemical staining for CyclinD1 and p21WAF1/Cip1, whereas p27Kip1 staining was reduced. Notably, spectral karyotyping showed that tumorigenic hMSC-TERT20 cells retained a normal diploid karyotype, with no detectable chromosome abnormalities. Consistent with the bone-forming potential of early passage hMSC-TERT20 cells, tumors derived from late passage cells expressed early biomarkers of osteogenesis. However, hMSC-TERT20 cells were heterogeneous for alpha smooth muscle actin (ASMA) expression and one out of six hMSC-TERT20 derived single cell clones was strongly ASMA positive. Tumors from this ASMA+ clone had distinctive vascular qualities with hot spots of high CD34+ murine endothelial cell density, together with CD34- regions with a branching periodic acid Schiff reaction pattern. Such clone-specific differences in host vascular response provide novel models to explore interactions between mesenchymal stem and endothelial cells. Despite the lack of a characteristic chromosomal translocation, the histomorphology, biomarkers and oncogenic changes were similar to those prevalent for Ewing's sarcomas. The phenotype and ontogenesis of hMSC-TERT20 tumors was consistent with the hypothesis that sarcomas may arise from hMSC, providing a unique diploid model for exploring human sarcoma biology.

Anxiety and repression in attention and retention.

High- and low-anxious college students (as determined by scores on the Taylor Manifest Anxiety Scale; A. W. Bendig, 1956) and repressors (low anxiety and high scores on the Marlowe-Crowne Social Desirability Scale; D. P. Crowne & D. Marlowe, 1964) were compared on 3 cognitive tasks. High-anxious participants more often spelled the negative emotional meaning of ambiguous homophones (e.g., pane/pain) and forgot more of their free associations to emotional cue words than did the low-anxious participants. The repressors also detected the emotional meaning of the homophones but unlike the anxious, the repressors did recall their associations to the emotional words. In a working memory task using nonemotional items, the moderately anxious participants recalled fewer words than did the low- and high-anxious participants. The results confirm that both trait anxiety and repression affect information processing at a variety of stages but not in the same way. Repressors were sensitive to, and retentive of, negative emotional stimuli.

Identifying the Ca(++) signalling sources activating chloride currents in Xenopus oocytes using ionomycin and thapsigargin.

The calcium ionophore, ionomycin (IM), and the sarcoplasmic/endoplasmic reticulum (SER) calcium pump inhibitor, thapsigargin (TG), were used to study the roles of Ca(++) from different sources in regulating Ca(++)-dependent Cl(-) currents in Xenopus oocytes. The Ca(++)-dependent Cl(-) currents, Ic, were measured in voltage-clamped oocytes (Vc = -60 mV). In the presence of extracellular Ca(++), both TG (0.1 to 10 microM) and IM (0.1 to 10 microM) induce release of Ca(++) from SER and activated capacitative Ca(++) entry (CCE) across the plasma membrane leading to activation of both "fast" and "slow" Cl(-) currents. The fast Ic was produced by Ca(++) release from SER while Ca(++) entry across the plasma membrane activated the slow Ic. Intracellular application of the calcium buffer, BAPTA, blocked activation of the slow Ic due to Ca(++) entry via CCE pathways, but not via IM-mediated movement across the plasma membrane. It is concluded that predominantly Ca(++) release from stores regulates a fast Ic while Ca(++) entry through CCE pathways regulates a slow Ic. Further, the CCE and slow Ic pathways must be located in spatially separated compartments since BAPTA can effectively abolish the effects of Ca(++) entry via the CCE pathway, but not by the IM-mediated entry pathway.

High level expression of differentially localized BAG-1 isoforms in some oestrogen receptor-positive human breast cancers.

Sensitivity to oestrogens and apoptosis are critical determinants of the development and progression of breast cancer and reflect closely linked pathways in breast epithelial cells. For example, induction of BCL-2 oncoprotein expression by oestrogen contributes to suppression of apoptosis and BCL-2 and oestrogen receptor (ER) are frequently co-expressed in tumours. BAG-1/HAP is a multifunctional protein which complexes with BCL-2 and steroid hormone receptors (including the ER), and can suppress apoptosis and influence steroid hormone-dependent transcription. Therefore, analysis of expression of BAG-1 in human breast cancer is of considerable interest. BAG-1 was readily detected by immunostaining in normal breast epithelial cells and most ER-positive tumours, but was undetectable or weakly expressed in ER-negative tumours. BAG-1 positive cells showed a predominantly cytoplasmic or cytoplasmic plus nuclear distribution of staining. A correlation between ER and BAG-1 was also evident in breast cancer derived cell lines, as all lines examined with functional ER expression also expressed high levels of BAG-1. In addition to the prototypical 36 kDa BAG-1 isoform, breast cancer cells expressed higher molecular weight isoforms and, in contrast to BCL-2, BAG-1 expression was independent of oestrogens. BAG-1 isoforms were differentially localized to the nucleus or cytoplasm and this was also independent of oestrogens. These results demonstrate a close association between BAG-1 and functional ER expression and suggest BAG-1 may be useful as a therapeutic target or prognostic marker in breast cancer.

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

Stepwise transformation of primary thyroid epithelial cells by a mutant Ha-ras oncogene: an in vitro model of tumor progression.

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.

Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T.

A recombinant murine retroviral vector encoding the SV40 virus large T antigen was used to infect stably an immortal line of differentiated rat thyroid epithelial cells, FRTL-5. Expression of SV40 T transformed these cells to anchorage independence and tumorigenicity but did not alter morphology or abolish tissue-specific functions and growth factor requirements. The resulting phenotype provides a model of well-differentiated human thyroid cancer.