Prognostic and therapeutic implications of mTORC1 and Rictor expression in human breast cancer.

The mammalian target of rapamycin (mTOR) plays a key role in the regulation of cellular metabolism, growth and proliferation. It forms two multi-protein complexes known as complex 1 (mTORC1) and 2 (mTORC2). Raptor and Rictor are the core proteins for mTORC1 and mTORC2, respectively. This study examines the relationship between mTORC1, Rictor and Raptor mRNA expression and human breast cancer. Furthermore, the correlation between mTORC1 and hTERT was investigated. Breast cancer tissues (n=150) and normal tissues (n=31) were analysed using reverse transcription and quantitative PCR. Transcript levels were correlated with clinicopathological data. Higher mTOR expression was noted in breast cancer tissue (P=0.0018), higher grade tumours (grade 2 vs. 3, P=0.047), in ductal tumours (P=0.0014), and was associated with worse overall survival (P=0.01). Rictor expression was significantly higher in background breast tissues compared with tumours and was inversely related to the Nottingham Prognostic Index (NPI1 vs. 2, P=0.03) and tumour grade (grade 1 vs. 3, P=0.01) and was associated with better overall (P=0.037) and disease-free survival (P=0.048). The mRNA expression of Raptor was higher in tumours compared with normal tissues. Furthermore, the expression of Raptor was associated with a higher tumour grade (grade 1 vs. 3, P=0.027). A highly significant positive correlation between mTOR and hTERT (P<0.00001) was observed. These observations are consistent with the role of mTORC1 in the anti-apoptosis pathway and suggest that selective inhibitors of mTORC1 may be more efficacious in human breast cancer. Our findings support the hypothesis that mTORC1 is an important upregulator of telomerase in breast cancer.

Carcinogen-specific mutational and epigenetic alterations in INK4A, INK4B and p53 tumour-suppressor genes drive induced senescence bypass in normal diploid mammalian cells.

Immortalization (senescence bypass) is a critical rate-limiting step in the malignant transformation of mammalian somatic cells. Human cells must breach at least two distinct senescence barriers to permit unfettered clonal evolution during cancer development: (1) stress- or oncogene-induced premature senescence (SIPS/OIS), mediated via the p16-Rb and/or ARF-p53-p21 tumour-suppressive pathways, and (2) replicative senescence triggered by telomere shortening. In contrast, because their telomerase is constitutively active, cells from small rodents possess only the SIPS/OIS barrier, and are therefore useful for studying SIPS/OIS bypass in isolation. Dermal fibroblasts from the Syrian hamster (SHD cells) are exceptionally resistant to spontaneous SIPS bypass, but it can be readily induced following exposure to a wide range of chemical and physical carcinogens. Here we show that a spectrum of carcinogen-specific mutational and epigenetic alterations involving the INK4A (p16), p53 and INK4B (p15) genes are associated with induced SIPS bypass. With ionizing radiation, immortalization is invariably accompanied by efficient biallelic deletion of the complete INK4/CDKN2 locus. In comparison, SHD cells immortalized by the powerful polycyclic hydrocarbon carcinogen benzo(a)pyrene display transversion point mutations in the DNA-binding domain of p53 coupled with INK4 alterations such as loss of expression of p15. Epimutational silencing of p16 is the primary event associated with immortalization by nickel, a human non-genotoxic carcinogen. As SIPS/OIS bypass is a prerequisite for the immortalization of normal diploid human epithelial cells, our results with the SHD model will provide a basis for delineating combinations of key molecular changes underpinning this important event in human carcinogenesis.

Prenatal exposure to diethylstilbestrol and long-term impact on the breast and reproductive tract in humans and mice.

The term 'developmental origins of health and disease' (DOHaD) originally referred to delayed effects of altered maternal factors (e.g. smoking or poor nutrition) on the developing offspring, but it now also encompasses early life exposure to environmental chemicals, which can cause an unhealthy prenatal environment that endangers the fetus and increases its susceptibility to disease later in life. Prenatal exposure to the pharmaceutical diethylstilbestrol (DES) is a well-known DOHaD example as it was associated in the 1970s with vaginal cancer in daughters who were exposed to this potent synthetic estrogen before birth. Subsequently, numerous long-term effects have been described in breast and reproductive tissues of DES-exposed humans and experimental animals. Data reviewed suggest that the prenatal DES-exposed population should continue to be monitored for potential-increased disease risks as they age. Knowledge of sensitive developmental periods, and the mechanisms of DES-induced toxicities, provides useful information in predicting potential adverse effects of other environmental estrogens.

Evidence for a tumour suppressor function of SETD2 in human breast cancer: a new hypothesis.

BACKGROUND: SET domain containing protein 2 (SETD2) is a histone methyltransferase that is involved in transcriptional elongation. We previously demonstrated SETD2 to be a potential tumour suppressor gene in breast cancer. The aim of this study was to compare SETD2 expression in breast cancer with that in adjacent non-cancerous breast tissue (ANCT) in paired samples. A hypothesis is proposed that explains the mode of action of SETD2 as a tumour suppressor gene. MATERIALS AND METHODS: Paired samples of tumour and adjacent non-cancerous tissue (ANCT) from 25 patients were analysed. The levels of transcription of SETD2 were determined using quantitative polymerase chain reaction and normalized against cytokeratin 19. Immunohistochemical staining with appropriate antibodies against SETD2 protein was also performed in selected samples. RESULTS: Levels of SETD2 mRNA were significantly higher in ANCT when compared to those in tumour samples (p=0.01). Immunohistochemistry also demonstrated a higher protein expression in ANCT. CONCLUSION: This study offers further evidence that SETD2 behaves like a tumour suppressor gene. Our hypothesis links SETD2 mode of action with telomerase regulation through human telomerase reverse transcriptase (hTERT). Several studies have emphasised the importance of histone methylation of hTERT promotor in telomerase regulation. SETD2 function of histone methylation could be the missing link in this chain which could explain the potential tumour suppressor function of SETD2.

Evidence of a tumour suppressor function for DLEC1 in human breast cancer.

UNLABELLED: DLEC1 (deleted in lung and oesophageal cancer), located on 3p22.3, is a candidate tumour suppressor gene in lung, esophageal, and renal cancer. The aim of this study was determine whether the mRNA expression levels of DLEC1 were consistent with a tumour suppressive function. MATERIALS AND METHODS: A total of 153 samples were analysed. The levels of transcription of DLEC1 were determined using quantitative PCR and normalised against (CK19). Transcript levels within breast cancer specimens were compared to normal background tissues. RESULTS: Levels of transcription were lower [corrected] in tumour samples compared to adjacent non cancerous tissue (ANCT) samples but this was not statistically significant (median 0.167 vs. 0.03; p=0.138). DLEC1 expression levels were significantly lower in samples from patients who developed metastasis, local recurrence, or died of breast cancer when compared to those who were disease free for >10 years (p=0.041). DISCUSSION: These findings are consistent with a possible tumour suppressor function of DLEC1 in breast cancer.

The mRNA expression of SETD2 in human breast cancer: correlation with clinico-pathological parameters.

BACKGROUND: SET domain containing protein 2 (SETD2) is a histone methyltransferase that is involved in transcriptional elongation. There is evidence that SETD2 interacts with p53 and selectively regulates its downstream genes. Therefore, it could be implicated in the process of carcinogenesis. Furthermore, this gene is located on the short arm of chromosome 3p and we previously demonstrated that the 3p21.31 region of chromosome 3 was associated with permanent growth arrest of breast cancer cells. This region includes closely related genes namely: MYL3, CCDC12, KIF9, KLHL18 and SETD2. Based on the biological function of these genes, SETD2 is the most likely gene to play a tumour suppressor role and explain our previous findings. Our objective was to determine, using quantitative PCR, whether the mRNA expression levels of SETD2 were consistent with a tumour suppressive function in breast cancer. This is the first study in the literature to examine the direct relationship between SETD2 and breast cancer. METHODS: A total of 153 samples were analysed. The levels of transcription of SETD2 were determined using quantitative PCR and normalized against (CK19). Transcript levels within breast cancer specimens were compared to normal background tissues and analyzed against conventional pathological parameters and clinical outcome over a 10 year follow-up period. RESULTS: The levels of SETD2 mRNA were significantly lower in malignant samples (p = 0.0345) and decreased with increasing tumour stage. SETD2 expression levels were significantly lower in samples from patients who developed metastasis, local recurrence, or died of breast cancer when compared to those who were disease free for > 10 years (p = 0.041). CONCLUSION: This study demonstrates a compelling trend for SETD2 transcription levels to be lower in cancerous tissues and in patients who developed progressive disease. These findings are consistent with a possible tumour suppressor function of this gene in breast cancer.

Evidence of a tumour suppressive function of E2F1 gene in human breast cancer.

BACKGROUND: The E2F family of transcription factors are key regulators of genes involved in cell cycle progression, cell fate determination, DNA damage repair and apoptosis. E2F1 is unique in that it contributes both to the control of cellular proliferation and cellular death. Furthermore, unlike other E2Fs, E2F1 responds to various cellular stresses. This study aimed to examine the level of mRNA expression of E2F1 gene in normal and malignant breast tissue and correlate the level of expression to tumour stage. MATERIALS AND METHODS: One hundred and twenty-seven breast cancer tissue and 33 normal tissues were analyzed. Levels of transcription of E2F1 were determined using real-time quantitative PCR, normalized against CK19. Levels of expression were analyzed against TNM stage, nodal involvement, tumour grade and distant metastasis. RESULTS: The levels of E2F1 mRNA were lower in malignant tissues. They declined further with increasing TNM stage. This became statistically significant when TNM stages 3 and 4 were compared to TNM stages 1 and 2 disease (TNM1 vs. TNM3 p = 0.032; TNM1 vs. TNM4 p = 0.032; TNM2 vs. TNM3 p = .019; TNM2 vs. TNM4 p = 0.021). The levels of E2F1 also fell with increasing tumour grade, when comparing grade 2 and 3 with grade 1, however, the differences were not statistically significant. CONCLUSION: These results are highly suggestive of the role of E2F1 as a tumour suppressive gene in human breast cancer.

Stimulatory and inhibitory effects of genistein on human uterine leiomyoma cell proliferation are influenced by the concentration.

BACKGROUND: Due to dietary exposure of women to genistein, a soy-derived phytoestrogen, and the estrogen responsiveness of uterine leiomyomas 'fibroids', we evaluated the effects of genistein (0.001-50 microg/ml) on human uterine leiomyoma (UtLM) cells versus uterine smooth muscle cells (UtSMCs) in vitro. METHODS: Light microscopy was used to determine the effects of genistein on cell morphology. Proliferation was assessed using a colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemistry. Flow cytometry was used to quantitate cells in the S-phase and those undergoing apoptosis. A fluorometric assay and confocal microscopy were used to detect caspase-3 activity and apoptotic bodies, respectively. RESULTS: In UtLM cells, low concentrations (< or = 1 microg/ml) of genistein stimulated proliferation, increased PCNA labeling and the percentage of cells in the S-phase, but this did not occur in UtSMCs. Higher concentrations (> or = 10 microg/ml) of genistein adversely affected the morphology, significantly inhibited proliferation, decreased PCNA labeling, increased caspase-3 activity and induced apoptosis in both cell types. CONCLUSIONS: Genistein's effects are concentration-dependent in both cell lines. Lower concentrations elicit proliferative effects on UtLM cells only; whereas, higher concentrations alter morphology, inhibit proliferation, and increase caspase activity and apoptosis in both cell types, with the latter two effects being more extensive in UtSMCs.

HTERT mRNA expression correlates with matrix metalloproteinase-1 and vascular endothelial growth factor expression in human breast cancer: a correlative study using RT-PCR.

BACKGROUND: Telomerase activity has been significantly associated with nodal metastasis and cellular proliferation in human breast cancer, indicating that its degree of expression has some form of vital control over the invasive nature of the malignancy concerned. Of the telomerase subunits, the reverse transcriptase (hTERT) is the main determinant of enzyme activity. Vascular endothelial growth factors (VEGF)-C and (VEGF)-D, matrix metalloprotease type 1 (MMP-1) and protease-activated receptors (PARs) have all been linked to promotion of tumour invasiveness and metastatic dissemination. This study aims to examine the association between hTERT transcription and that of VEGF-D, VEGF-C, MMP-1, PAR1a and PAR1b through a correlative analysis of the mRNA transcripts of these genes in human breast cancer. MATERIALS AND METHODS: Breast cancer tissues (n = 116) and normal tissues (n-31) were collected immediately after surgery and stored at -80 degrees C until use. The level of hTERT transcripts from the prepared DNA from the above samples was determined using real time-quantitative PCR based on the Amplifluor technology. The levels of the transcript were generated from a standard that was simultaneously amplified with the samples. Normalisation against cytokeratin 19 (CK19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also carried out. RESULTS: There was a positive correlation between hTERT mRNA expression (after CK19 normalisation) with both VEGF-D and MMP-1 in human breast cancer. PAR1 was seen to correlate with hTERT (after GAPDH normalisation) with a highly significant correlation with PAR1a alone. However there was no correlation between hTERT transcription and VEGF-C or with PAR1b alone. CONCLUSION: Our findings suggest that hTERT is a potential up-regulator of MMP-1, PAR1 and VEGF-D expression and this may explain its apparent control over the invasiveness and metastasis of the malignancy concerned.

Contribution of BRCA1 and BRCA2 germline mutations to the incidence of early-onset breast cancer in Cyprus.

In Cyprus, the prevalence of breast cancer associated with BRCA1 and BRCA2 mutations in young women is unknown. In this study, we present the results of mutational analysis of the BRCA1 and BRCA2 genes in 26 Cypriot women diagnosed with breast cancer by the age of 40. The entire coding regions, including splice sites, of the BRCA1 and BRCA2 genes were sequenced using cycle sequencing. We identified four pathogenic mutations: two in BRCA1 [c.1840A>T (K614X), c.5310delG (5429delG)] and two in BRCA2 [c.3531-3534delCAGC (3758del4), c.8755delG (8984delG)] in six of 26 unrelated patients. The BRCA2 mutation c.3531-3534delCAGC (3758del4) is novel and the BRCA1 mutation c.1840A>T (K614X) is reported for the first time in Cypriot patients. The BRCA2 Cypriot founder mutation c.8755delG (8984delG) was detected in three unrelated patients. Additionally, we identified one novel BRCA1 missense mutation, two novel polymorphisms and three novel intronic variants of which BRCA1 c.4185+3A>G (IVS12+3A>G) may be pathogenic. Of the six BRCA1/2 mutation carriers, only four had a family history. These results show that the prevalence of BRCA1 and BRCA2 mutations in Cypriot women diagnosed with early-onset breast cancer is high. We conclude that Cypriot women with early-onset breast cancer should be offered BRCA1/2 testing irrespective of their family history.

hTERT mRNA expression is associated with a poor clinical outcome in human breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens including the Nottingham Prognostic Index (NPI) and to explore the relationship between hTERT expression and clinical outcome. MATERIALS AND METHODS: RNA was extracted from 116 breast carcinomas and 31 matched adjacent non-cancerous tissue (ANCT). hTERT mRNA expression was estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. RESULTS: hTERT mRNA was present in all of the cancerous specimens (mean=0.1701, median=0.0205) and most ANCT specimens with levels being 2.6 times higher in the cancerous tissue than in ANCT (mean=0.156 vs. 0.68, p=0.18). The mean mRNA levels increased with NPI scores (0.0816 for NPI 1, 0.1186 for NPI 2 and 0.68 for NPI 3), however this failed to reach statistical significance (P-values= 0.33 for NPI 1 vs. 2, 0.27for NPI2 vs. 3 and 0.24 for NPI 1 vs. 3). hTERT levels also increased with increasing tumour's grade (mean= 0.0459 for grade 1, 0.111for grade 2, and 0.27 for grade 3) but this trend did not reach a statistical significance. Low levels of hTERT were associated with mucinous carcinoma compared with ductal (p=0.023) and lobular (p=0.021) types. hTERT mRNA levels were higher in patients who had recurrent disease or died from breast cancer compared with those who remained alive without disease after a median follow up of 6 years (p=0.0026). CONCLUSION: High hTERT mRNA levels are associated with a poor clinical outcome in human breast cancer and should be included as a prognostic marker in future validation studies.

hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. MATERIALS AND METHODS: The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. RESULTS: hTERT expression was positive in 27 (71%) of the 38 tumours. 15 (79%) of 19 node positive tumours were hTERT positive compared with 11 (63%) of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388). There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097). CONCLUSION: hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer.

Evidence for a tumour suppressive function of APRG1 in breast cancer.

INTRODUCTION: Deletion mapping studies have shown that genes in the region 3p21.3 are often deleted in epithelial malignancies. Although the regions deleted differ between individual cancers there appears to be a specificity for the type of malignancy produced. As a result this area of chromosome 3 is thought to contain multiple tumour suppressor genes. The present study concentrates on one gene in that region, APRG1. This gene codes for a protein, which has been implicated in cell membrane interactions. The aim was to determine using quantitative PCR whether the levels of this gene were negatively correlated with clinical outcome in breast cancer. METHODS: One hundred and twenty tumour tissues and 33 normal tissues were analyzed. Levels of transcription of APRG1 were determined using real-time quantitative PCR. APRG1 expression was normalized against CK19. Levels of expression were analyzed against staging, nodal involvement, grade, distant metastasis and survival over a 6 year follow up period. RESULTS: Levels of APRG1 mRNA were lower in malignant tissues. They fell further with increasing stage using the TNM classification This became statistically significant when TNM stages 3 and 4 were compared to TNM 1 (p = 0.0046, p = 0.04, t-test). They were lower in those with positive nodes although this did not reach statistical significance. There was a statistically significant reduction in APRG1 in grade 3 tumours cf. grade 1 (p = 0.0081). APRG1 expression was highly negatively correlated with progressive disease: alive with metastasis (p = 0.0069), local recurrence (p = 0.0055), died of breast cancer (p = 0.11), all progressive disease (p = 0.035). CONCLUSION: This study shows a compelling trend for APRG1 transcription levels to be lower in malignant tissues and lower still in those patients who develop progressive disease. There was also a statistically significant difference in APRG1 levels between grade 3 and grade 1 tumours. These results are highly suggestive of APRG1 acting as a tumour suppressor gene.

Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid.

The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 A. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).

Long term dietary methoxychlor exposure in rats increases sodium solution consumption but has few effects on other sexually dimorphic behaviors.

Methoxychlor is an insecticide with estrogen-like activity, thus exposure during development might cause sexually dimorphic behavioral alterations. To evaluate this, pregnant rats consumed diets containing 0, 10, 100 or 1000 ppm methoxychlor from gestational day 7, and offspring continued on these diets until postnatal day (PND) 77. Assessments of sexually dimorphic behaviors in offspring indicated that intake of a 3.0% sodium chloride solution was significantly increased (41%) in males and females of the 1000 ppm group. No treatment group differed from controls in open field nor running wheel activity, play behavior, nor 0.3% saccharin solution intake. Offspring of the 1000 ppm group showed significantly decreased body weight, reaching 17% less than controls at PND 77, but not clearly related to their salt solution intake. During pregnancy, 1000 ppm dams consumed 23% less food and weighed 10% less than controls, but this did not affect litter outcomes. These results indicate that in rodents, developmental and chronic exposure to dietary methoxychlor alters the sexually dimorphic behavior of salt-solution intake in young adults of both sexes. Similar behavioral alterations with other xenoestrogens, and the potential for interactions among xenoestrogens, suggest that this report may minimize the true effects of dietary methoxychlor exposure.

Myelotoxicity in genistein-, nonylphenol-, methoxychlor-, vinclozolin- or ethinyl estradiol-exposed F1 generations of Sprague-Dawley rats following developmental and adult exposures.

The myelotoxicity of five endocrine active chemicals was evaluated in F1 generation of Sprague-Dawley rats following developmental and adult exposures at three concentration levels. Rats were exposed to genistein (GEN: 25, 250 and 1250 ppm), nonylphenol (NPH: 25, 500 and 2000 ppm), methoxychlor (MXC: 10, 100 and 1000 ppm), vinclozolin (VCZ: 10, 150 and 750 ppm) and ethinyl estradiol (EE2: 5, 25 and 200 ppb) gestationally and lactationally through dams from day 7 of gestation and through feed after weaning on postnatal day (PND) 22 to PND 64. The parameters examined included the number of recovered bone marrow cells, DNA synthesis, and colony forming units (CFU) in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin. Except for the EE2, the concentrations of other individual chemicals in the diet were in an approximate range that allowed for a comparison to be made in terms of myelotoxic potency. Decreases in the DNA synthesis, CFU-GM and CFU-M seemed to be the common findings among the alterations induced by these compounds. Using the numbers of alterations induced by each chemical in the parameters examined as criteria for comparison, the order of myelotoxic potency in F(1) males was: GEN>MXC>NPH>VCZ; the order in females: GEN>NPH>VCZ. Additionally, some of the functional changes induced by these compounds were gender-specific or dimorphic. Overall, the results demonstrated that developmental and adult exposures of F1 rats to these endocrine active chemicals at the concentrations tested had varied degrees of myelotoxicity with GEN being the most potent. Furthermore, the sex-specific effects of these chemicals in F1 male and female rats suggest that there may be interactions between these compounds and sex hormone in modulating these responses.

WNT5A expression in human breast cancer.

UNLABELLED: The Wnt family encodes secreted signaling molecules involved in cell adhesion and, by implication, cell growth. Wnt5a has been shown to behave as a putative oncogene and also as a tumour suppressor gene. This is a reflection of its role within a multi-step pathway and in the variety of ways in which its production can be stimulated or switched off. Wnt genes can be functionally separated into two classes; those that activate the canonical Wnt/beta-catenin pathway and those that activate the Wnt/Ca++ pathway. Wnt5a signals through frizzled receptors and, depending upon which frizzled receptor is present, may activate either pathway. Therefore the observed function of Wnt5a is entirely dependent upon its context, hence the confusion over its role in tumorigenesis. This study examines Wnt5a mRNA expression using RT-PCR in human breast cancer. MATERIALS AND METHODS: One hundred and twenty malignant breast tumours and 33 normal breast tissues were analysed. The levels of transcription of Wnt5a were determined using real-time quantitative PCR. Levels of expression were analysed against staging, nodal involvement, grade, distant metastasis and survival over a 6-year follow-up period. RESULTS: Levels of Wnt5a mRNA were lower in tumours than in normal tissue (mean values: 107 vs. 62.7). They fell further with increasing stage using the Nottingham Prognostic Index. This became statistically significant when NPI3 was compared to normal tissue (p=0.043, t-test). There was a trend towards lower levels of Wnt5a in those with progressive disease, however, this did not reach statistical significance. In patients with ER-negative disease, lower levels of Wnt5a were significantly associated with a worse clinical outcome (p=0.016). CONCLUSION: There is a trend for mRNA levels to be lower in cancerous tissue and lower still in those showing more aggressive behaviour. This is consistent with the hypothesis that Wnt5a is a tumour suppressor gene with potential clinical applications.

Dietary methoxychlor exposure modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in F0 and F1 generations of Sprague Dawley rats.

Methoxychlor, a chlorinated hydrocarbon pesticide, is a persistent environmental contaminant that has been identified in human reproductive tissues. Methoxychlor has been shown to be estrogenic in both in vivo and in vitro studies. As an endocrine disrupter, it may have the potential to adversely affect endocrine, reproductive, and immune systems in animals. The present study evaluated methoxychlor's immunotoxic potential in F0 (dams) and F1 generations of Sprague Dawley rats exposed to an isoflavone-free diet containing methoxychlor at concentrations of 10, 100, and 1000 ppm. In dams, exposure to methoxychlor from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the NK activity (1000 ppm) and the percentages of T cells (1000 ppm), helper T cells (1000 ppm) and macrophages (100 and 1000 ppm). In contrast, a decrease in the numbers of splenocytes and B cells was observed at the 100 and 1000 ppm concentrations. In F1 males, exposure to methoxychlor gestationally, lactationally and through feed from postnatal day 22-64 (78 days total exposure) produced an increase in the spleen IgM antibody-forming cell response to sheep red blood cells (100 and 1000 ppm) and the activity of NK cells (1000 ppm). However, there was a decrease in the terminal body weight (1000 ppm), spleen weight (1000 ppm), thymus weight (100 and 1000 ppm), and the numbers of splenocytes (1000 ppm), B cells (100 and 1000 ppm), cytotoxic T cells (1000 ppm) and NK cells (100 and 1000 ppm). In F1 females, exposure to methoxychlor produced a decrease in the terminal body weight (1000 ppm) and the percentages of cytotoxic T cells (10, 100 and 1000 ppm). These results demonstrate that developmental and adult dietary exposure to methoxychlor modulates immune responses in Sprague Dawley rats. Immunological changes were more pronounced in the F1 generation male rats that were exposed during gestation and postpartum, when compared to the F0 and F1 generation females. Increases in antibody-forming cell response and NK cell activity, and altered spleen cell subpopulation numbers were observed in the F1 generation male rats, without similar changes to the F1 generation females.

Increased volume of the calbindin D28k-labeled sexually dimorphic hypothalamus in genistein and nonylphenol-treated male rats.

The adult rat brain develops through an interplay of neuronal proliferation and programmed cell death. Steroid hormones and growth factors may alter the balance between these competing processes. "Endocrine disrupters" (EDs) may also alter brain development, by mimicry or modulation of endogenous hormone systems. Under control conditions, the sexually dimorphic nucleus (SDN) of the medial preoptic hypothalamus becomes larger in adult males than females, but its final volume may also reflect the hormonal conditions prevailing during development. Two EDs that have recently been studied in protocols involving lifespan exposures are the phytoestrogen genistein and the weakly estrogenic compound para-nonylphenol, which is used in the production of many surfactants and plastics. Experimental dietary exposure of adult female rats to genistein or p-nonylphenol began 28 days prior to their mating at concentrations of 5 ppm, 100 ppm, and 500 ppm for genistein or 25 ppm, 200 ppm, and 750 ppm for p-nonylphenol. Exposure of the offspring continued throughout gestation and lactation, as well as in their chow after weaning, until they were sacrificed at 140 days of age for immunohistochemical labeling of the calbindin D28k-labeled subdivision of the SDN: the CALB-SDN. Both genistein and nonylphenol were found to increase the volume of the CALB-SDN in male rats (p's < 0.01), but not in female rats.