|
| 1. |
Gallmeier, E., Hucl, T., Brody, J.R., Dezentje, D.A., Tahir, K., Kasparkova, J., Brabec, V., Bachman, K.E. and Kern, S.E.
(2007)
High-throughput screening identifies novel agents eliciting hypersensitivity in Fanconi pathway-deficient cancer cells.
Cancer Res.
67
,
2169–2177
.
|
| |
Notes:
The Fanconi anemia (FA) pathway is inactivated in a variety of human tumors. Identifying novel compounds that affect FA-pathway deficient cells could provide further information on the FA pathway as well as new therapeutic possibilities. In this study, RKO cells, a human cancer cell line stably expressing firefly luciferase under the control of the p53 consensus DNA-binding site, were treated with 10 and 20µmol/L 80136342, a new compound that enhances cancer cell survival, or 50µmol/L etoposide, a known activator of p53, for 24 hours. The reporter activity was assessed using the Steady-Glo® Luciferase Assay System and compared to that seen in untreated controls.
(0003600) |
| |
 |
| |
Products: Steady-Glo® Luciferase Assay System |
| 2. |
Narita, K., Chien, J., Mullany, S.A., Staub, J., Qian, X., Lingle, W.L. and Shridhar, V.
(2007)
Loss of HSulf-1 expression enhances autocrine signaling mediated by amphiregulin in breast cancer.
J. Biol. Chem.
March 15
,
Epub (ahead of print)
.
|
| |
Notes:
These authors identified HSulf-1 as a downregulated gene in ovarian, breast and several other cancer cell lines. To investigate the clinical impact of this downregulation, tissue microarray was used look for associations between HSulf-1 expression levels and clinico-pathological parameters such as tumor histology, grade, hormone receptor status and presence of recurrent disease. HSulf-1 expression levels were determined by RNA in situ hybridization, using probes developed with the Riboprobe® System.
(0003616) |
| |
|
| |
Products: Riboprobe® System—SP6 |
| 3. |
Curtis, C.D., Likhite, V.S., McLeod, I.X., Yates, J.R. and Nardulli, A.M.
(2007)
Interaction of the tumor metastasis suppressor nonmetastatic protein 23 homologue H1 and estrogen receptor alpha alters estrogen-responsive gene expression.
Cancer Res.
67
,
10600–10607
.
|
| |
Notes:
Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR® Green dye.
(0003789) |
| |
|
| |
Products: Reverse Transcription System |
| 4. |
Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.
(2007)
Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells.
Mol. Cell. Endocrinol.
264
,
50-60
.
|
| |
Notes:
This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms.
(0003618) |
| |
|
| |
Products: Caspase-Glo® 3/7 Assay | CellTiter-Blue® Cell Viability Assay | ImProm-II™ Reverse Transcriptase | M-MLV Reverse Transcriptase Buffer Pack | M-MLV Reverse Transcriptase, RNase H Minus | pTARGET™ Mammalian Expression Vector System |
| 5. |
Garcia-Pedrero, J.M, Kiskinis, E., Parker, M.G., and Belandia, B.
(2007)
The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.
J. Biol. Chem.
281
,
22656–22664
.
|
| |
Notes:
To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with 35S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQueous One Solution Cell Proliferation Assay.
(0003599) |
| |
|
| |
Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pRL-CMV Vector |
| 6. |
Dong, M., How, T., Kirkbride, K.C., Gordon, K.J., Lee, J.D., Hempel, N., Kelly, P., Moeller, B.J., Marks, J.R., and Blobe, G.C.
(2007)
The type III TGF-b receptor suppresses breast cancer progression.
J. Clin. Invest.
117
,
206-17
.
|
| |
Notes:
These authors showed that loss of the type II TGF-β receptor TGFβIII through allelic imbalance occurs during breast cancer development and increases metastatic potential. When TGFβIII expression was restored in human breast cancer cells, invasiveness, angiogenesis and metastasis were inhibited in an in vivo model system. The authors first analyzed TGFβIII mRNA levels using a cDNA array of 50 different breast cancer samples and controls. They also investigated TGFβIII protein expression levels by immunohistochemical analysis of a breast cancer tissue array containing over 250 breast cancers specimens at different stages of disease progression. Results from both analyses showed that TGFβIII expression decreased as disease progressed. The effect pf TGFβIII expression on tumor growth was investigated in a mouse model system. Murine 4T1 mammary cancer cells genetically engineered to express firefly luciferase were stably transfected with an expression vector containing TGFβIII , or a control vector. Tumor progression was then monitored in vivo by bioluminescent imaging. Cells expressing TGFβIII had delayed onset of metastasis and a reduction on the size and number of metastases compared with non-TGFβIII-expressing cells.
(0003617) |
| |
|
| |
Products: Luciferin-EF™ |
| 7. |
Adams, B.D., Furneaux, H. and White, B.
(2007)
The micro-RNA miR-206 targets the human estrogen receptor-α, and represses ERα mRNA and protein expression in breast cancer cells.
Mol. Endocrinol.
Mar. 13
,
Epub (ahead of print)
.
|
| |
Notes:
This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells.
(0003603) |
| |
|
| |
Products: Luciferase Assay System |
| 8. |
Xu, H., Shan, J., Jurukovski, V., Yuan, L., Li, J. and Tian, K.
(2007)
TSP50 encodes a testis-specific protease and is negatively regulated by p53.
Cancer Res.
67
,
1239–1245
.
|
| |
Notes:
TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo® Assay System.
(0003598) |
| |
|
| |
Products: Beta-Glo® Assay System | Luciferase Assay Reagent | Luciferase Assay System | pGL3-Basic Vector | pGL3-Enhancer Vector |
| 9. |
Ghosh, S., Lu, Y., Katz, A., Hu, Y., and Li, R.
(2007)
Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells.
Am. J. Physiol. Endocrinol. Metab.
292
,
E246-E252
.
|
| |
Notes:
Obesity-associated elevated estrogen increases the risk for breast cancer in postmenopausal women. The rate limiting step in the synthesis of estrogen from androgen is catalyzed by the aromatase enzyme. Normally this enzyme is expressed under a weak promoter in adipose tissue; however in breast cancer a second, strong ovary-specific promoter (PII) drives expression of aromatase. This study investigated the relationship of BRCA1 and aromatase expression. RNA isolated from BRCA-1 siRNA-treated adipose stromal cells was reverse transcribed using the ImProm-II™ Reverse Transcription System. The authors show that siRNA knockdown of BRCA1 resulted in activation of the PII promoter, suggesting that BRCA1 can modulate estrogen biosynthesis in adipose tissue.
(0003606) |
| |
|
| |
Products: ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System |
| 10. |
Cheng, G.Z., Chan, J., Wang, Q., Zhang, W. and Wang, L-H.
(2007)
Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion and resistance to paclitaxel.
Cancer Res.
67
,
1979-1987
.
|
| |
Notes:
To study the molecular mechanism underlying metatasis, the authors established a model system to select highly invasive cells. Increased Twist and AKT2 expression was noted in the highly invasive cells and the authors sought a functional connection between these two proteins. HEK293T cells were transfected with AKT2-Luc, a control vector encoding Renilla luciferase, and increasing amounts of Myc-Twist. The Dual-Luciferase® Reporter Assay System was used to measure firefly luciferase activity, normalized to Renilla activity to determine whether Twist was able to transactivate full-length AKT2 promoter. The results, as measured by luciferase activity, showed that Twist led to a dosage-dependent increase in AKT2 promoter transactivation.
(0003605) |
| |
|
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 11. |
Trauernicht, A.M., Kim, S.J., Kim, N.H., and Boyer, T.G.
(2007)
Modulation of estrogen receptor alpha protein level and survival function by DBC-1.
Mol. Endocrinol.
21
,
1526-1536
.
|
| |
Notes:
These authors used a proteomics-based approach to isolate and identify proteins that interact with the estrogen receptor ERα. The protein DBC-1 was identified as a ligand-independent binding partner of ERα. The amino terminus of DBC-1 bound directly to the ERα hormone binding domain in the absence of estradiol. Initial identification of DBC-1 was based on peptide sequence analysis of proteins that coimmunoprecipitated with unliganded ERα. The CheckMate™ Mammalian Two-Hybrid System was then used to confirm the interaction. DBC-1 was fused to the GAL4-DNA-binding domain in the pBIND vector, and ERα was fused to the VP16 activation domain in the pACT vector. These proteins were then expressed independently or together in HeLa cells and their ability to activate transcription of a luciferase reporter controlled by GAL-4-DNA binding sites (pG5luc) was evaluated in both the presence and absence of estradiol. Further analysis with truncated proteins was used to demonstrate that the interaction between DBC-1 and ERα was mediated by the amino terminal part of the DBC-1 protein.
(0003628) |
| |
|
| |
Products: CheckMate™ Mammalian Two-Hybrid System |
| 12. |
Chen, E.I., Hewel, J., Kreuger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R., and Felding-Habermann, B.
(2007)
Adaptation of energy metabolism in breast cancer brain metastases.
Cancer Res.
67
,
1472-1486
.
|
| |
Notes:
This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis.
(0003613) |
| |
|
| |
Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 13. |
Muraoka-Cook, R.S., Caskey, L.S., Sandahl, M.A., Hunter, D.M., Husted, C., Strunk, K.E., Sartor, C.I., Rearick, W.A., McCall, W., Sgagias, M.K., Cowan, K.H., and Earp, H. S.
(2007)
Heregulin-dependent delay in mitotic progression requires HER4 and BRCA1.
Mol. Cell. Biol.
26
,
6412-6424
.
|
| |
Notes:
Heregulin is a growth factor that binds to the HER/ErbB family of receptors, which includes four HER members. HER1 and HER2 overexpression in breast cancer cells is associated with higher malignancy and poorer prognosis. However, HER4 expression is associated with longer survival and more positive prognosis. The authors of this study investigated HER4-dependent growth inhibition that is mediated by heregulin. The authors determined the absolute levels of HER4 mRNA in several human breast cancer and mouse cancer cell lines. For this determination, relative cell numbers were determined using a CellTiter® AQueous MTS assay. Apoptosis in heregulin-treated or untreated cells was also determined using the DeadEnd™ Colorimetric TUNEL System. The authors concluded that heregulin decreases growth of HER4-positive breast cancer cells, and that this growth inhibition is dependent on BRCA1.
(0003596) |
| |
|
| |
Products: CellTiter 96® AQueous MTS Reagent Powder | CellTiter 96® AQueous One Solution Cell Proliferation Assay | DeadEnd™ Colorimetric TUNEL System | Olomoucine (cdc2 Protein Kinase Inhibitor) |
| 14. |
Chrestensen, C.A., Shuman, J.K., Eschenroeder, A., Worthington, M., Gram, H. and Sturgill, T.W.
(2007)
MNK1 and MNK2 regulation in HER2-overexpressing breast cancer lines.
J. Biol. Chem.
282
,
4243–4252
.
|
| |
Notes:
These authors investigated the regulation and function of MAPK-interacting protein kinases 1 and 2 (MNK 1 and 2) and other proteins involved in the MAPK pathway in a panel of breast cancer cells. They use an anti-phospho-ERK antibody produced by Promega against a proprietary immunogen to examine the activation status and amounts of ERKs by Western blot analysis.
(0003609) |
| |
|
| |
|
| 15. |
Ma, Y., Katiyar, P., Jones, L.P., Fan, S., Zhang, Y., Furth, P.A., and Rosen, E.M.
(2006)
The breast cancer susceptibility gene BRCA1 regulates progesterone receptor signaling in mammary epithelial cells.
Molecular Endocrinology
20
,
14-34
.
|
| |
Notes:
The authors of this study investigate the relationship between BRCA1 and activity of the progesterone receptor (PR) in mammary tumor cells. BRCA1 mutations confer increased risk for steroid hormone-responsive cancers such as endometrial and cervical cancers and prostate cancer. In this study, evidence for interaction between PR and BRCA1 is presented. Glutathione-S-transferase (GST) capture assays were used to determine if PR and BRCA1 interact directly. GST-PR fusion proteins are used to pull down in vitro transcribed and translated BRCA1. In vitro transcription and translation reactions were carried out using a TNT® Rabbit Reticulocyte Lysate system. Interaction between BRCA1 and PR isoforms A and B was observed, and this interaction did not appear to require the presence of progesterone. The authors also showed that BRCA1 regulates expression of several progesterone-responsive genes.
(0003602) |
| |
|
| |
Products: TNT® SP6 Coupled Reticulocyte Lysate System | TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size | TNT® T7/SP6 Coupled Reticulocyte Lysate System | TNT® T7/T3 Coupled Reticulocyte Lysate System |
| 16. |
Holzscheiter, L., Biermann, J.C., Kotzsch, M., Prezas P., Farthmann, J., Baretton, G., Luther, T., Tjan-Heijnen, V.C.G., Taliere, M., Schmitt, M., Sweep, F.C.G.J., Span, P.N. and Magdolen, V.
(2006)
Quantitative reverse transcription-pcr assay for detection of mRNA encoding full-length human tissue kallikrein 7: Prognostic relevance of KLK7 mRNA expression in breast cancer.
Clin. Chem.
52
,
1070-1079
.
|
| |
Notes:
Few reliable prognostic markers have been developed for breast cancer. Human tissue kallikreins (hKs) are serine proteases with diverse functions. Fifteen human tissue kallikrein genes (KLK) have been identified. Several human tissue kallikreins have been associated with malignancies, such as hK3 (prostate-specific antigen) and prostate cancer. hK7 has been linked to a significantly poorer prognosis for ovarian and breast cancer, indicating that it may serve as a marker for these diseases. However, previous analyses of KLK7 expression were non-selective studies of all KLK7 mRNA forms. The authors developed a quantitative reverse-transcription-PCR (QPCR) assay, using the Reverse Transcription System on RNA, then developed a highly sensitive QPCR assay, to determine whether a more specific analysis of full-length KLK7 mRNA might lead to similar results as those from previous studies on the multiple KLK7 mRNA forms, with the ultimate goal of determining whether KLK7 could serve as a marker for breast cancer.
(0003615) |
| |
|
| |
Products: Reverse Transcription System |
| 17. |
Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.
(2006)
MicroRNA regulates the expression of human cytochrome P450 1B1.
Cancer Res.
66
,
9090-9098
.
|
| |
Notes:
These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells.
(0003622) |
| |
|
| |
Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | P450-Glo™ CYP1B1 Assay | pGL3-Promoter Vector | pTARGET™ Mammalian Expression Vector System |
| 18. |
Gritsko, T., Williams, A., Turkson, J., Kaneko, S., Bowman, T., Huang, M., Nam, S., Eweis, I., Diaz, N., Sullivan, D., Yoder, S., Enkemann, S., Eschrich, S., Lee, J.H., Beam, C.A., Cheng, J., Minton, S., Muro-Cacho, C.A. and Jove, R.
(2006)
Persistent activation of stat3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells.
Clin. Cancer Res.
12
,
11–19
.
|
| |
Notes:
These authors identified survivin as a Stat3-regulated gene in breast cancer cells using microarray analysis. The authors used a pGL2 construct containing the survivin gene promoter driving firefly luciferase expression and a Promega luciferase assay system to monitor survivin promoter activity in NIH 3T3 cells. Cotransfection with vectors expressing Stat3 and v-src, which activates endogenous Stat3, increased survivin promoter activity by almost fivefold, whereas expression of a dominant-negative Stat3 variant decreased survivin promoter activity by 50%.
(0003610) |
| |
|
| |
Products: pGL2-Basic Vector |
| 19. |
Udayakumar, T.S., Belakavadi, M., Choi, K.H., Pandey, P.K. and Fondell, J.D.
(2006)
Regulation of Aurora-A kinase gene expression via GABP recruitment of TRAP220/MED1.
J. Biol. Chem.
281
,
14691–14699
.
|
| |
Notes:
TRAP220/MED1 is amplified in estrogen receptor-positive breast cancer cells and has been shown to interact with a number of transcription factors essential for cell growth and development including BRCA-1 and p53. TRAP220/MED1 is a subunit of the TRAP/Mediator coactivator complex. These authors used RNA interference to reduce TRAP220/MED1 expression by >90%, then microarray analysis to identify genes that were downregulated after TRAP220/MED1 depletion. One such gene was Aurora-A serine/threonine kinase. The authors created Aurora-A-firefly luciferase constructs to determine the effect of TRAP220/MED1 depletion on Aurora-A promoter activity. As a positive control, the authors used a thyroid hormone (T3)-responsive firefly luciferase construct to show that depletion of TRAP220/MED1, which is known to play a role in nuclear receptor-mediated gene activation, interferes with thyroid hormone receptor-mediated activation of T3-responsive genes. Luciferase reporter gene activity was measured using the Dual Luciferase Reporter Assay System, and results were normalized to Renilla luciferase expression from the pRL-TK Vector.
(0003607) |
| |
|
| |
Products: Dual-Luciferase® Reporter Assay System | pRL-TK Vector |
| 20. |
Budhram-Mahadeo, V.S., Bowen, S., Lee, S., Perez-Sanchez, C., Ensor, E., Morris, P.J. and Latchman, D.S.
(2006)
Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression.
Nucleic Acids Res.
34
,
6640–6652
.
|
| |
Notes:
Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with 35S-methionine labeled full-length or truncated p53, prepared using the TNT® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase® Reporter Assay System.
(0003597) |
| |
|
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL4.74[hRluc/TK] Vector | pRL-TK Vector | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size |
| 21. |
Shukla, V., Coumoul, X., Cao, L., Wang, R.-H., Xiao, C., Xu, X., Andò, S., Yakar, S., LeRoith, D. and Deng, C.
(2006)
Absence of the full-length breast cancer-associated gene-1 leads to increased expression of insulin-like growth factor signaling axis members.
Cancer Res.
66
,
7151-7157
.
|
| |
Notes:
The authors of this study investigated the influence of BRCA1 on insulin-like growth factor 1 (IGF-1) signaling. In mice lacking full-length BRCA, they observed increased IGF-1 expression as well as changes in expression of other proteins within the IGF-1 signaling pathway including Irs-I. They observed increases in IGF-I and Irs-I expression in mammary tumors from these same mice. To understand better the relationship between BRCA1 and IGF, they transfected a mouse mammary tumor cell line with a small hairpin RNA directed against BRCA1 and showed that Irs-1 mRNA and promoter activity increases. Similar results were observed in human UBR60 cells. Irs-1 promoter activity was assessed using the Dual-Luciferase® Reporter Assay System.
(0003604) |
| |
|
| |
Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 22. |
McCabe, N., Turner, N.C., Lord, C.J., Kluzek, K., Bialkowska, A., Swift, S., Giavara, S., O'Connor, M.J., Tutt, A.N., Zdzienicka, M.Z., Smith, G.C.M., and Ashworth, A.
(2006)
Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-Ribose) polymerase inhibition.
Cancer Res.
66
,
8109-8115
.
|
| |
Notes:
The authors of this study investigated the basis of PARP inhibition sensitivity of cells containing mutations in BRCA1 or BRCA2 and whether the role of BRCA1 and 2 in homologous recombination might underlie the PARP inhibition sensitivity. They transfected HeLa cells with antisense constructs targeted against several proteins involved in homologous recombination and assessed sensitivity to PARP inhibition. Cells containing the RNAi constructs were treated with PARP inhibitors and blasticidin and cell viability was measured using the CellTiter-Glo® Cell Viability Assay. Antisense inhibition of several genes including those coding for RAD51, replication protein A1 and checkpoint kinase 2 among others, resulted in sensitivity to PARP inhibition. These results indicate that the role of BRCA1 and BRCA2 in homologous recombination contributes to the sensitivity to PARP inhibition.
(0003595) |
| |
|
| |
Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 23. |
Moyano, J.V., Evans, J.R., Chen, F., Lu, M., Werner, M.E., Yehiely, F., Diaz, L.K., Turbin, D., Karaca, G., Wiley, E., Nielsen, T.O., Perou, C.M. and Cryns, V.L.
(2006)
αB-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer.
J. Clin. Invest.
116
,
261-270
.
|
| |
Notes:
While exploring existing breast cancer cDNA microarrays the authors noted that alpha-basic-crystallin (αB-crystallin) was frequently expressed in basal-type breast carcinomas and wondered if αB-crystallin might contribute to the aggressive nature of these types of breast cancer. To identify how αB-crystallin disrupts mammary acinar architecture, the analyzed MCF-10A cell pools, grown in 1% horse serum (no added EGF) for activation of signaling pathways key to cell transformation. They used the CellTiter 96® AQueous One Solution Cell Proliferation Assay to examine cell viability at various days, compared to day 0. The authors observed that MCF-10A-αB-WT cells expressed higher levels of total and phosphorylated ERK1/2, Akt and p38 than did non-αB-containing parental cell types.
(0003614) |
| |
|
| |
Products: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay |
| 24. |
Chen, I-F., Ou-Yang, F., Hung, J-Y., Liu, J-C., Wang, H., Wang, S-C., Hou, M-F., Hortobagyi, G. and Hung, M-C.
(2006)
AIM2 suppresses human breast cancer cell proliferation in vitro and mammary tumor growth in a mouse model.
Mol. Cancer Ther.
5
,
1-7
.
|
| |
Notes:
The authors aimed to determine whether AIM2 inhibits breast cancer cell growth in vitro and explored the possibility of using AIM2 in breast cancer gene therapy. They generated tetracycline-inducible AIM2 cells lines, using pBI-EGFP-luc plasmid as a control. MCF-7 Tet-Off cells (human breast cancer cells) were transfected with pBI-EGFP-Tag-AIM2 or pBI-EGFP-Luc using SN liposome. Two days later cells were selected with blasticidin and tetracycline-derivative doxycycline. Blasticidin-resistant cells were screened for induction of GFP by fluorescence microscopy or for AIM2 expression (Western blotting) after removal of doxycycline. Luciferase activity induction was confirmed using the Dual-Luciferase® Reporter Assay System. Two AIM2 expressing stable cells lines were then selected and used for subsequent experiments, in vitro, to examine whether whether expression of AIM2 could suppress breast cancer cell proliferation and tumor formation.
(0003608) |
| |
|
| |
|