G1P3, an interferon- and estrogen-induced survival protein contributes to hyperplasia, tamoxifen resistance and poor outcomes in breast cancer.

Hormonally regulated survival factors can have an important role in breast cancer. Here we elucidate G1P3, a survival protein induced by interferons (IFNs), as a target of estrogen signaling and a contributor to poor outcomes in estrogen receptor-positive (ER(+)) breast cancer. Compared with normal breast tissue, G1P3 was upregulated in the malignant epithelium (50 × higher) and was induced by estrogen ex vivo. In accord with its overexpression in early stages of breast cancer (hyperplasia and ductal carcinoma in situ), in morphogenesis assays G1P3 enhanced the survival of MCF10A acinar luminal cells causing hyperplasia by suppressing detachment-induced loss of mitochondrial potential and apoptosis (anoikis). In cells undergoing anoikis, G1P3 attenuated the induction of Bim protein, a proapoptotic member of the Bcl-2 family and reversed the downmodulation of Bcl-2 protein. Downregulation of G1P3 induced spontaneous apoptosis in BT-549 breast cancer cells and significantly reduced the growth of ER(+) breast cancer cell MCF7 (P≤0.01), further suggesting its prosurvival activity. In agreement with its induction by estrogen, G1P3 antagonized tamoxifen, an inhibitor of ER in MCF7 cells. More importantly, elevated expression of G1P3 was significantly associated with decreased relapse-free and overall survival in ER(+) breast cancer patients (P≤0.01). Our studies suggest that elevated expression of G1P3 may perturb canonical tumor-suppressing activity of IFNs partly by affecting the balance of pro- and antiapoptotic members of Bcl-2 family proteins, leading to breast cancer development and resistance to therapies.

Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL.

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.

Quality of hospital care in African American and white patients with ischemic stroke and TIA.

OBJECTIVE: To examine whether differences exist in the in-hospital diagnostic evaluation and treatment of African American and white patients with ischemic stroke (IS) and TIA. METHODS: The authors used a state-wide hospital-based stroke registry prototype designed to measure and track the quality of acute stroke care. Weighted descriptive statistics for each racial group are reported for the following variables, which were deemed to be potential confounders of the association between race and the quality of stroke care: age, gender, insurance status, emergency medical services arrival, functional status on presentation, modified Rankin score at discharge, stroke subtype, neurologist involved in care, and stroke pathway utilization. The magnitude and significance of the associations between race and each quality indicator of in-hospital acute stroke care were determined by separate multiple logistic regression models, adjusting for all potential confounding variables. RESULTS: Among patients admitted with IS and TIA who were alive at discharge (n = 1,837), 340 (18.5%) were African American and 1497 (81.5%) were white. After multivariate analysis, African Americans were less likely to have a door-to-CT time of less than 25 minutes (odds ratio [OR] 0.13 [CI 0.049 to 0.32]), obtain cardiac monitoring (OR 0.54 [CI 0.29 to 1.03]), undergo dysphagia screening (OR 0.69 [CI 0.50 to 0.95]), and receive smoking cessation counseling (OR 0.27 [CI 0.17 to 0.42]). CONCLUSIONS: Quality of hospital care for African American and white patients with acute ischemic stroke and TIA was similar in many respects. However, African Americans were less likely to receive a CT within 25 minutes of arrival, cardiac monitoring, dysphagia screening, and smoking cessation counseling.

IV tissue plasminogen activator use in acute stroke: experience from a statewide registry.

OBJECTIVE: To assess the use of IV recombinant tissue plasminogen activator (rt-PA) in a statewide hospital-based stroke registry and to identify factors associated with its use among eligible patients. METHODS: A modified stratified sampling scheme was used to obtain a representative sample of 16 hospitals. Prospective case ascertainment and data collection were used to identify all acute stroke admissions over a 6-month period. Subjects eligible for IV rt-PA were defined as those who arrived within 3 hours of onset, who had no evidence of hemorrhage on initial brain image, and who had no physician-documented reasons for non-treatment with IV rt-PA. Multivariate logistic regression was used to identify factors associated with IV rt-PA use. RESULTS: Of 2,566 stroke admissions, 330 (12.9%) met the eligibility criteria for rt-PA treatment, and of these 43 (13%) received IV rt-PA treatment. Among 2,236 admissions excluded from consideration, 21% had evidence of hemorrhage on initial imaging, 35% had unknown stroke onset times, 38% had an onset to arrival time >3 hours, and 6% had physician documented contraindications. Among eligible patients, being male, use of emergency medical services, and rapid presentation were associated with increased IV rt-PA use. CONCLUSIONS: Treatment with IV rt-PA was underutilized in this hospital-based stroke registry. The primary reason for nontreatment was delayed presentation. Reducing prehospital and in-hospital response times would help increase IV rt-PA use, as would greater emergency medical services use. Improving the documentation of onset times would help clarify the underlying causes of delayed presentation.

Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis.

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway.

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.

In vivo expression of the interleukin 4 receptor alpha by astrocytes in epilepsy cerebral cortex.

We reported previously that non-neoplastic astrocytes (derived from brain tissues of patients with epilepsy) expressed interleukin 4 receptor alpha (IL-4Ralpha) and responded to interleukin 4 (IL-4) in culture. To determine whether reactivity of cultured astrocytes was relevant to primary tissue, we investigated IL-4Ralpha expression in specimens of non-neoplastic cerebral cortex removed for surgical treatment of intractable epilepsy compared to specimens of glial tumours, which have been reported to contain IL-4Ralpha. Freshly frozen tissues from eight cases (four epilepsy, four malignant astrocytoma) were evaluated for IL-4Ralpha expression by reverse-transcriptase polymerase chain reaction (RT-PCR), Southern blotting, and double-labelled immunohistochemistry with antibodies to IL-4Ralpha and glial fibrillary acidic protein (GFAP). IL-4Ralpha mRNA was detectable in both non-neoplastic and neoplastic tissues, whereas interleukin 2 receptor gamma chain (IL-2Rgammac) mRNA was not found. By immunohistochemistry, IL-4Ralpha protein co-localized to cells displaying GFAP and astrocytic morphology in epilepsy tissues. As anticipated, IL-4Ralpha was detectable in astrocytoma, but, surprisingly, was also observed in GFAP-positive, non-neoplastic "reactive" astrocytes adjacent to tumour. Results are consistent with the concept that non-neoplastic epilepsy astrocytes express IL-4Ralpha in situ, thus confirming in vitro studies and implying IL-4 sensitivity in vivo.

Interleukin-13 sensitivity and receptor phenotypes of human glial cell lines: non-neoplastic glia and low-grade astrocytoma differ from malignant glioma.

Many of the actions and receptor components of interleukin-13 (IL-13), a pleiotrophic cytokine with immunotherapeutic potential, are shared with IL-4. Because human low-grade astrocytoma cells express IL-4 receptors and their growth is arrested by IL-4, we speculated that IL-13 sensitivity and receptor expression might also be present. The purpose of the current study was to investigate IL-13 receptor components and sensitivity in a series of glial cell lines derived from adult human non-neoplastic cerebral cortex, low-grade astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme. Unlike peripheral blood lymphocytes (PBL), glial cells did not express IL-2 receptor gamma chain. IL-13 receptor alpha-1 (IL-13Ralpha1), however, was present in 11/13 glial lines and PBL. Deficient cell lines were all glioblastoma-derived. All anaplastic astrocytoma and glioblastoma but not other glial lines or PBL expressed IL-13 receptor alpha-2 (IL-13Ralpha2). In non-neoplastic glia, low-grade, and anaplastic astrocytoma, IL-13 decreased DNA synthesis, an effect reversible with antibody to IL-4Ralpha. Results indicate that low-grade astrocytoma cells resemble non-neoplastic glia in terms of IL-13 sensitivity and IL-4Ralpha/IL-13Ralpha1 receptor profile but alterations occur with malignant progression. Glioblastoma cells were uniformly insensitive to IL-13 and, unlike other glia, failed to phosphorylate STAT6 after IL-13 challenge. Data suggest that IL-13 and analysis of IL-13 receptors may have clinical application in glial tumors.

Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells.

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.

Homocysteine as a risk factor for ischemic stroke: an epidemiological story in evolution.

Homocysteine is a metabolite of methionine that may be remethylated by enzymes requiring folate and cobalamin (vitamin B12) to again form methionine or catabolized by the pyridoxine (vitamin B6) dependent enzyme, cystathionine beta synthase (CBS) to form cysteine (fig. 1) [1]. Homocysteine exists as a combination of various free and protein bound forms, but the total amount is what is usually measured and may be reported as homocyst(e)ine [2]. The biological plausibility that elevated homocysteine might lead to vascular disease noted in 1969 by McCully [3]. He reported that a child with abnormal cobalamin metabolism and hyperhomocysteinemia had arterial lesions similar to those seen in children with severe hyperhomocysteinemia from CBS deficiency. These findings led to the idea that moderate elevations in homocysteine, even those still within the so-called normal range, might also lead to vascular pathology through a variety of mechanisms including atherosclerosis and thrombosis [4].

Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts.

One of the main functions of mammalian peroxisomes is the beta-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal beta-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the beta-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Interleukin-4 down-regulates adult human astrocyte DNA synthesis and proliferation.

The T lymphocyte-derived cytokine, interleukin-4 (IL-4), was found to inhibit dose dependently basal DNA synthesis of cultured non-neoplastic human astrocytes isolated from epilepsy white matter tissue. The mitogenic effect of tumor necrosis factor on astrocytes was also inhibited by IL-4, and the inhibitory effect was abrogated by anti-IL-4 antibody but not by irrelevant IgG. Immunofluorescent analysis indicated significantly reduced numbers of glial fibrillary acidic protein-positive astrocytes incorporating nuclear bromodeoxyuridine in IL-4-treated cultures compared to control. These findings indicate that human adult astrocyte proliferation, in contrast to that reported for endothelial cells or fibroblasts, is sensitive to down-regulation by IL-4.

Divergent responses of human astrocytoma and non-neoplastic astrocytes to tumor necrosis factor alpha involve the 55 kDa tumor necrosis factor receptor.

The effects of tumor necrosis factor (TNF) on DNA synthesis, proliferation, and induction of gene/protein expression of TNF were compared in neoplastic and non-neoplastic adult human astrocytes. Previously, we demonstrated that TNF induced proliferative responses in non-neoplastic adult human astrocytes. In astrocytoma cells, however, TNF elicited both proliferative and cytostatic responses depending upon cell density and TNF concentration. This bimodal effect persisted even in a homogeneous, cloned astrocytoma cell line (STT-9C), and was inhibitable by neutralizing antibody to TNF. TNF treatment enhanced expression of TNF mRNA in astrocytoma cells but not in non-neoplastic astrocytes, and cell-associated or secreted TNF was detectable in any culture. The involvement of receptors in astrocyte responses to TNF was examined in serological studies using monoclonal antibodies Utr-1 to the 75 kDa, and Htr-9 to the 55 kDa TNF receptor. Antibody to the 55 kDa TNF receptor alone was able to mimic the effects of TNF in both neoplastic and non-neoplastic astrocyte cultures while antibody to the 75 kDa TNF receptor had no effect. These data indicate that the bimodal actions of TNF on human astrocytoma cells as well as the stimulatory effects on non-neoplastic adult astrocytes are regulated at least in part by the 55 kDa TNF receptor. Astrocyte TNF receptors, however, do not appear to constitute part of an autocrine growth pathway in either non-neoplastic or neoplastic human astrocytes.

Human astrocytes proliferate in response to tumor necrosis factor alpha.

Two different human astrocytic cell lines derived from adult epilepsy surgical specimens were exposed in vitro to concentrations of 1-100 ng/ml recombinant tumor necrosis factor alpha (TNF alpha). Results indicated dose-dependent stimulation of DNA synthesis and proliferation. Both of these effects were abrogated by treatment with monoclonal antibody specific for TNF alpha but not by irrelevant murine IgG. Immunocytochemical characterization of TNF alpha-treated and control cultures indicated that greater than 98% of proliferating cells contained cytoplasmic glial fibrillary acidic protein (GFAP), and were therefore astrocytic in nature. These studies demonstrate that growth of adult human non-neoplastic astrocytes is stimulated by TNF alpha, an inflammatory cytokine produced primarily by macrophages but also by astrocytes.

Characterization of adult human astrocytes derived from explant culture.

Four different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP(+) cells also proliferated, bromo-deoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material.

Birth order and sex of sibling as determinants of mother-infant interaction.

Mother-infant interaction was assessed on 32 first- and second-born siblings when each was 3 months old. Data were colleted during 2 6-hour naturalistic home observations using a modified time-sampling technique. The sample consisted of 4 equal-size subgroups of same and opposite sex sibling pairs. Results suggested that interaction between a mother and her infant varied depending on the birth order and gender of the infant. Mothers spent significantly less time in social, affectionate, and caretaking interaction (except for feeding activities) with their second borns than they had with their firstborns; this difference was greater if the second born was female. Certain patterns of maternal behaviors appeared to be stable from one sibling to the other. Different types of interaction between the mothers and their younger infants were related to attention-seeking behavior in the firstborn male and female siblings.