P53 regulation of osteoblast differentiation is mediated through specific microRNAs.

In order to understand the role of the p53 tumor suppressor gene in microRNA expression during osteoblast differentiation, we used a screen to identify microRNAs that were altered in a p53-dependent manner. MicroRNAs from MC3T3-E1 preosteoblasts were isolated from day 0 (undifferentiated) and day 4 (differentiating) and compared to a p53 deficient MC3T3-E1 line treated similarly. Overall, one fourth of all the microRNAs tested showed a reduction of 0.6 fold, and a similar number of them were increased 1.7 fold with differentiation. P53 deficiency caused 40% reduction in expression of microRNAs in differentiating cells, while a small percent (0.03%) showed an increase. Changes in microRNAs were validated using real-time PCR and two microRNAs were selected for further analysis (miR-34b and miR-140). These two microRNAs were increased significantly during differentiation but showed a dramatic reduction in expression in a p53 deficient state. Stable expression of miR-34b and miR-140 in MC3T3-E1 cells resulted in decreases in cell proliferation rates when compared to control cells. There was a 4-fold increase in p53 levels with miR-34b expression and a less dramatic increase with miR-140. Putative target binding sites for bone specific transcription factors, Runx2 and Osterix, were found for miR-34b, while Runx2, beta catenin and type 1 collagen were found to be miR-140 targets. Western blot analyses and functional assays for the transcription factors Runx2, Osterix and Beta-catenin confirmed microRNA specific interactions. These studies provide evidence that p53 mediated regulation of osteoblast differentiation can also occur through specific microRNAs such as miR-34b and miR-140 that also directly target important bone specific genes.

Cadmium Nephrotoxicity Is Associated with Altered MicroRNA Expression in the Rat Renal Cortex.

Cadmium (Cd) is a nephrotoxic environmental pollutant that causes a generalized dysfunction of the proximal tubule characterized by polyuria and proteinuria. Even though the effects of Cd on the kidney have been well-characterized, the molecular mechanisms underlying these effects have not been fully elucidated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate cellular and physiologic function by modulating gene expression at the post-transcriptional level. The goal of the present study was to determine if Cd affects renal cortex miRNA expression in a well-established animal model of Cd-induced kidney injury. Male Sprague-Dawley rats were treated with subcutaneous injections of either isotonic saline or CdCl2 (0.6 mg/kg) 5 days a week for 12 weeks. The 12-week Cd-treatment protocol resulted in kidney injury as determined by the development of polyuria and proteinuria, and a significant increase in the urinary biomarkers Kim-1, ß2 microglobulin and cystatin C. Total RNA was isolated from the renal cortex of the saline control and Cd treated animals, and differentially expressed miRNAs were identified using µParafloTM microRNA microarray analysis. The microarray results demonstrated that the expression of 44 miRNAs were significantly increased and 54 miRNAs were significantly decreased in the Cd treatment group versus the saline control (t-test, p ≤ 0.05, N = 6 per group). miR-21-5p, miR-34a-5p, miR-146b-5p, miR-149-3p, miR-224-5p, miR-451-5p, miR-1949, miR-3084a-3p, and miR-3084c-3p demonstrated more abundant expression and a significant two-fold or greater increased expression in the Cd-treatment group versus the saline control group. miR-193b-3p, miR-455-3p, and miR-342-3p demonstrated more abundant expression and a significant two-fold or greater decreased expression in the Cd-treatment group versus the saline control group. Real-time PCR validation demonstrated (1) a significant (t-test, p ≤ 0.05, N = 6 per group) increase in expression in the Cd-treated group for miR-21-5p (2.7-fold), miR-34a-5p (10.8-fold), miR-146b-5p (2-fold), miR-224-5p (10.2-fold), miR-3084a-3p (2.4-fold), and miR-3084c-3p (3.3-fold) and (2) a significant (t-test, p ≤ 0.05, N = 6 per group) 52% decrease in miR-455-3p expression in the Cd-treatment group. These findings demonstrate that Cd significantly alters the miRNA expression profile in the renal cortex and raises the possibility that dysregulated miRNA expression may play a role in the pathophysiology of Cd-induced kidney injury. In addition, these findings raise the possibility that Cd-dysregulated miRNAs might be used as urinary biomarkers of Cd exposure or Cd-induced kidney injury.

Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA, and MK-5108 on SW-872 and 93T449 human liposarcoma cells.

Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0-1000 nM), AZD1152-HQPA (0-1000 nM), and AMG 900 (0-1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC50 = 3.7 nM) > AZD1152-HQPA (EC50 = 43.4 nM) > MK-5108 (EC50 = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC50 = 6.5 nM) > AZD1152-HQPA (EC50 = 74.5 nM) > MK-5108 (EC50 = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0-1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Effects of cadmium on the sub-cellular localization of ß-catenin and ß-catenin-regulated gene expression in NRK-52E cells.

The E-cadherin/ß-catenin complex is a structural component of adherens-type junctions in epithelial cells. Moreover, ß-catenin acts as an intracellular signaling molecule that can influence the expression of a variety of genes that regulate apoptosis and cell cycle control. Cadmium (Cd) is an environmental toxicant that causes renal dysfunction and disrupts cadherin-dependent cell-cell adhesion in various types of epithelial cells. In this study, we examined the effects of Cd on the subcellular localization of ß-catenin, the cadherin/ß-catenin complex and ß-catenin-mediated gene transcription in rat proximal tubule NRK-52E cells. Exposure to 5-10 µM Cd for 4 h caused the NRK cells to separate from each other without killing the cells or causing them to detach from the growing surface. This effect was associated with the loss of ß-catenin and E-cadherin from the cell-cell contacts and apparent changes in the accumulation of ß-catenin in the nuclear cell subfraction. The expression of the ß-catenin-sensitive gene, c-jun was significantly increased in cells exposed to 5 µM Cd. However, there was no change in the expression of several other ß-catenin-regulated genes including: c-myc, cyclin D1 and matrilysin. Additional studies utilizing the TOPFLASH ß-catenin reporter gene construct showed that Cd caused a 2-3 fold increase in the expression of the luciferase reporter gene. Overall, these results indicate that Cd disrupts the cadherin/ß-catenin complex in NRK-52E cells, but this effect leads to only partial activation of ß-catenin-mediated gene transcription.

Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5.

The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All-trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 (ankyrin repeat SOCS box 2) gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins, Cul5, regulator of cullin 2 (ROC2), and elongin B and C. The purpose of this study was to determine if there is increased expression of Cul5 during granulocytic differentiation of HL-60 cells. To induce granulocytic differentiation, HL-60 cells were treated for 5 d with ATRA and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time RT-PCR was used to measure Cul5 mRNA expression and also the expression of other components of the E3 ubiquitin ligase (ASB-2, ROC2, elongin B and C). Granulocytic differentiation of HL-60 cells was associated with a 1.6-, 1.7-, and 23-fold statistically significant (P

Differential effects of domoic acid and E. coli lipopolysaccharide on tumor necrosis factor-alpha, transforming growth factor-beta1 and matrix metalloproteinase-9 release by rat neonatal microglia: evaluation of the direct activation hypothesis.

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

Analysis of CUL-5 expression in breast epithelial cells, breast cancer cell lines, normal tissues and tumor tissues.

BACKGROUND: The chromosomal location of CUL-5 (11q 22-23) is associated with LOH in breast cancer, suggesting that CUL-5 may be a tumor suppressor. The purpose of this research was to determine if there is differential expression of CUL-5 in breast epithelial cells versus breast cancer cell lines, and normal human tissues versus human tumors. The expression of CUL-5 in breast epithelial cells (HMEC, MCF-10A), and breast cancer cells (MCF-7, MDA-MB-231) was examined using RT-PCR, Northern blot analysis, and Western blot analysis. The expression of mRNA for other CUL family members (CUL-1, -2, -3, -4A, and -4B) in these cells was evaluated by RT-PCR. A normal human tissue expression array and a cancer profiling array were used to examine CUL-5 expression in normal human tissues and matched normal tissues versus tumor tissues, respectively. RESULTS: CUL-5 is expressed at the mRNA and protein levels by breast epithelial cells (HMEC, MCF-10A) and breast cancer cells (MCF-7, MDA-MB-231). These cells also express mRNA for other CUL family members. The normal human tissue expression array revealed that CUL-5 is widely expressed. The cancer profiling array revealed that 82% (41/50) of the breast cancers demonstrated a decrease in CUL-5 expression versus the matched normal tissue. For the 50 cases of matched breast tissue there was a statistically significant approximately 2.2 fold decreased expression of CUL-5 in tumor tissue versus normal tissue (P < 0.0001). CONCLUSIONS: The data demonstrate no apparent decrease in CUL-5 expression in the breast cancer cell lines (MCF-7, MDA-MB-231) versus the breast epithelial cells (HMEC, MCF-10A). The decrease in CUL-5 expression in breast tumor tissue versus matched normal tissue supports the hypothesis that decreased expression of CUL-5 may play a role in breast tumorigenesis.

Immunohistochemical evaluation of vasopressin expression in breast fibrocystic disease and ductal carcinoma in situ (DCIS).

We previously found that expression of the vasopressin gene is a common feature of human breast cancer. In the present study we first examined 21 different cases of benign fibrocystic breast disease for vasopressin expression using immunohistochemistry and antibodies directed against vasopressin (anti-VP) and against vasopressin-associated glycopeptide (anti-VAG). All cases examined were negative for vasopressin gene expression using these antibodies. Alternatively, we examined 16 cases of breast ductal carcinoma in situ (DCIS) using the second of these antibodies (anti-VAG), and all of these cases were positive for vasopressin gene expression. Our results suggest that products of vasopressin gene expression are not markers of cellular proliferation in the breast, and might rather represent an early part of the carcinogenic process in this tissue.

Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequesters beta-catenin in human cervical epithelial cells.

The cadherin/catenin complex serves as an important structural component of adherens junctions in epithelial cells. Under certain conditions, beta-catenin can be released from this complex and interact with transcription factors in the nucleus to stimulate the expression of genes that regulate apoptosis and cell cycle control. While studying the effects of the bacterial pathogen Chlamydia trachomatis on human cervical epithelial cells in culture, we observed that C. trachomatis caused the epithelial cells to separate from each other without detaching from their growing surface. The objective of the present study was to determine if this effect might involve the disruption of the cadherin/catenin complex. Primary cultures of human cervical epithelial cells or HeLa cells were infected with C. trachomatis serovar E. Cadherin-like immunoreactive materials and beta-catenin were visualized by immunofluorescence. Preliminary studies showed that N-cadherin was the primary cadherin expressed in both the primary cultures and the HeLa cells. In noninfected cells, N-cadherin and beta-catenin were colocalized at the intercellular junctional complexes. By contrast, the infected cells showed a marked loss of both N-cadherin and beta-catenin labeling from the junctional complexes and the concomitant appearance of intense beta-catenin labeling associated with the chlamydial inclusion. The results of Western blot analyses of extracts of C. trachomatis showed no evidence of cross-reactivity with the beta-catenin antibody. These results indicate that C. trachomatis causes the breakdown of the N-cadherin/beta-catenin complex and that the organism can sequester beta-catenin within the chlamydial inclusion. This could represent an important mechanism by which C. trachomatis alters epithelial cell function.