Novel roles for GAPDH in cell death and carcinogenesis.
Artículo
Tuesday, December 01, 2009
Sunday, November 08, 2009
A selective eradication of human non-hereditary breast cancer cells by phenanthridine derived polyADP-ribose polymerase inhibitors
Dana Inbar-Rozensal email, Asher Castiel email, Leonid Visochek email, David Castel email, Francoise Dantzer email, Shai Izraeli email and Malka Cohen-Armon email
Breast Cancer Research 2009, 11:R78doi:10.1186/bcr2445
Published: 5 November 2009
Abstract (provisional)
Introduction
PARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells.
Methods
In-vitro (cell cultures) and in vivo (xenotransplants) experiments were performed.
Results
PARP inhibition by phenanthridine derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human non-hereditary breast cancer cells MCF-7 and MDA231. However, while the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with phenanthridine derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors.
Conclusions
Breast Cancer Research 2009, 11:R78doi:10.1186/bcr2445
Published: 5 November 2009
Abstract (provisional)
Introduction
PARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells.
Methods
In-vitro (cell cultures) and in vivo (xenotransplants) experiments were performed.
Results
PARP inhibition by phenanthridine derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human non-hereditary breast cancer cells MCF-7 and MDA231. However, while the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with phenanthridine derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors.
Conclusions
p53 responsive elements in human retrotransposons
Oncogene (2009) 28, 3857–3865; doi:10.1038/onc.2009.246; published online 31 August 2009
C R Harris1,2, A DeWan3, A Zupnick4, R Normart1, A Gabriel5, C Prives4, A J Levine6 and J Hoh3
Monday, October 26, 2009
Pten in stromal fibroblasts suppresses mammary epithelial tumours. Nature. 2009 Oct 22;461(7267):1084-91.
Trimboli AJ, Cantemir-Stone CZ, Li F, Wallace JA, Merchant A, Creasap N, Thompson JC, Caserta E, Wang H, Chong JL, Naidu S, Wei G, Sharma SM, Stephens JA, Fernandez SA, Gurcan MN, Weinstein MB, Barsky SH, Yee L, Rosol TJ, Stromberg PC, Robinson ML, Pepin F, Hallett M, Park M, Ostrowski MC, Leone G.
Tumor Microenvironment Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA
The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.
Resto de las Figuras (Libre acceso)
http://www.nature.com/nature/journal/v461/n7267/fig_tab/nature08486_ft.html
Tuesday, October 20, 2009
Distinct inherited metastasis susceptibility exists for different breast cancer subtypes: a prognosis study
Szu-Min Hsieh 
Breast Cancer Research 2009, 11:R75doi:10.1186/bcr2412
| Published: | 13 October 2009 |
Abstract (provisional)
Introduction
Previous studies in mouse models and pilot epidemiology studies have demonstrated that inherited polymorphisms are associated with inherited risk of tumor progression and poor outcome in human breast cancer. To extend these studies and gain better understanding of the function of inherited polymorphism in breast cancer progression a validation prognosis study was performed in a large independent breast cancer patient population.
Methods
The study population consisted of 1863 Dutch patients with operable primary breast cancer from Rotterdam, The Netherlands. Genomic DNA was genotyped for the missense Pro436Leu RRP1B single nucleotide polymorphism (SNP) rs9306160 and the intronic SIPA1 SNP rs2448490 by SNP-specific PCR.
Results
A significant association of variants in RRP1B with metastasis-free survival was observed (P = 0.012), validating the role of RRP1B with inherited metastatic susceptibility. Stratification of patients revealed that association with patients' survival was found to be specifically restricted to estrogen receptor positive, lymph node-negative (ER+/LN-) patients (P = 0.011). The specific association with metastasis-free survival only in ER+/LN- patients was replicated for SIPA1, a second metastasis susceptibility gene known to physically interact with RRP1B (P = 0.006). Combining the genotypes of these two genes resulted in the significant ability to discriminate patients with poor metastasis-free survival (HR: 0.40, 95% CI: 0.24 to 0.68, P = 0.001).
Conclusions
These results validate SIPA1 and RRP1B as metastasis susceptibility genes and suggest that genotyping assays may be a useful supplement to other clinical and molecular indicators of prognosis. The results also suggest that lymphatic and hematogeneous metastases are genetically distinct that may involve different mechanisms. If true, these results suggest that metastatic disease, like primary breast cancer, may be multiple diseases and that stratification of late stage patients may therefore be required to fully understand breast cancer progression and metastasis.
Tuesday, October 06, 2009
IgG nobel prizes awarded
On October 1, the 2009 IgNobel awards were handed out at Harvard University. The winners:
VETERINARY MEDICINE PRIZE: Catherine Douglas and Peter Rowlinson of Newcastle University, Newcastle-Upon-Tyne, UK, for showing that cows who have names give more milk than cows that are nameless.
PEACE PRIZE: Stephan Bolliger, Steffen Ross, Lars Oesterhelweg, Michael Thali and Beat Kneubuehl of the University of Bern, Switzerland, for determining — by experiment — whether it is better to be smashed over the head with a full bottle of beer or with an empty bottle.
ECONOMICS PRIZE: The directors, executives, and auditors of four Icelandic banks — Kaupthing Bank, Landsbanki, Glitnir Bank, and Central Bank of Iceland — for demonstrating that tiny banks can be rapidly transformed into huge banks, and vice versa — and for demonstrating that similar things can be done to an entire national economy.
CHEMISTRY PRIZE: Javier Morales, Miguel Apátiga, and Victor M. Castaño of Universidad Nacional Autónoma de México, for creating diamonds from liquid — specifically from tequila.
MEDICINE PRIZE: Donald L. Unger, of Thousand Oaks, California, USA, for investigating a possible cause of arthritis of the fingers, by diligently cracking the knuckles of his left hand — but never cracking the knuckles of his right hand — every day for more than sixty (60) years.
PHYSICS PRIZE: Katherine K. Whitcome of the University of Cincinnati, USA, Daniel E. Lieberman of Harvard University, USA, and Liza J. Shapiro of the University of Texas, USA, for analytically determining why pregnant women don't tip over.
LITERATURE PRIZE: Ireland's police service (An Garda Siochana), for writing and presenting more than fifty traffic tickets to the most frequent driving offender in the country — Prawo Jazdy — whose name in Polish means "Driving License".
PUBLIC HEALTH PRIZE: Elena N. Bodnar, Raphael C. Lee, and Sandra Marijan of Chicago, Illinois, USA, for inventing a brassiere that, in an emergency, can be quickly converted into a pair of protective face masks, one for the brassiere wearer and one to be given to some needy bystander.
MATHEMATICS PRIZE: Gideon Gono, governor of Zimbabwe’s Reserve Bank, for giving people a simple, everyday way to cope with a wide range of numbers — from very small to very big — by having his bank print bank notes with denominations ranging from one cent ($.01) to one hundred trillion dollars ($100,000,000,000,000).
BIOLOGY PRIZE: Fumiaki Taguchi, Song Guofu, and Zhang Guanglei of Kitasato University Graduate School of Medical Sciences in Sagamihara, Japan, for demonstrating that kitchen refuse can be reduced more than 90% in mass by using bacteria extracted from the feces of giant pandas.
An interesting and entertaining crop as always. Links to papers and other information can be found here.
VETERINARY MEDICINE PRIZE: Catherine Douglas and Peter Rowlinson of Newcastle University, Newcastle-Upon-Tyne, UK, for showing that cows who have names give more milk than cows that are nameless.
PEACE PRIZE: Stephan Bolliger, Steffen Ross, Lars Oesterhelweg, Michael Thali and Beat Kneubuehl of the University of Bern, Switzerland, for determining — by experiment — whether it is better to be smashed over the head with a full bottle of beer or with an empty bottle.
ECONOMICS PRIZE: The directors, executives, and auditors of four Icelandic banks — Kaupthing Bank, Landsbanki, Glitnir Bank, and Central Bank of Iceland — for demonstrating that tiny banks can be rapidly transformed into huge banks, and vice versa — and for demonstrating that similar things can be done to an entire national economy.
CHEMISTRY PRIZE: Javier Morales, Miguel Apátiga, and Victor M. Castaño of Universidad Nacional Autónoma de México, for creating diamonds from liquid — specifically from tequila.
MEDICINE PRIZE: Donald L. Unger, of Thousand Oaks, California, USA, for investigating a possible cause of arthritis of the fingers, by diligently cracking the knuckles of his left hand — but never cracking the knuckles of his right hand — every day for more than sixty (60) years.
PHYSICS PRIZE: Katherine K. Whitcome of the University of Cincinnati, USA, Daniel E. Lieberman of Harvard University, USA, and Liza J. Shapiro of the University of Texas, USA, for analytically determining why pregnant women don't tip over.
LITERATURE PRIZE: Ireland's police service (An Garda Siochana), for writing and presenting more than fifty traffic tickets to the most frequent driving offender in the country — Prawo Jazdy — whose name in Polish means "Driving License".
PUBLIC HEALTH PRIZE: Elena N. Bodnar, Raphael C. Lee, and Sandra Marijan of Chicago, Illinois, USA, for inventing a brassiere that, in an emergency, can be quickly converted into a pair of protective face masks, one for the brassiere wearer and one to be given to some needy bystander.
MATHEMATICS PRIZE: Gideon Gono, governor of Zimbabwe’s Reserve Bank, for giving people a simple, everyday way to cope with a wide range of numbers — from very small to very big — by having his bank print bank notes with denominations ranging from one cent ($.01) to one hundred trillion dollars ($100,000,000,000,000).
BIOLOGY PRIZE: Fumiaki Taguchi, Song Guofu, and Zhang Guanglei of Kitasato University Graduate School of Medical Sciences in Sagamihara, Japan, for demonstrating that kitchen refuse can be reduced more than 90% in mass by using bacteria extracted from the feces of giant pandas.
An interesting and entertaining crop as always. Links to papers and other information can be found here.
Monday, October 05, 2009
´Técnica para identificar mRNAs que se traducen versus mRNA aquellos que no se están traduciendo.
*4E-BP Extends Lifespan upon Dietary Restriction by Enhancing Mitochondrial Activity in Drosophila. Cell, Volume 139, Issue 1, 149-160, 2 October 2009. Zid et al.
En este paper utilizan un sistema para separar mRNAs que se están traduciendo activamente versus aquellos mRNAs que se traducen poco y aquellos que se no están traduciendo (esto mediante gradientes asumiendo que los muy activos tienen muchos ribosomas versus aquellos no activos carecen de ribosomas) después hicieron microarreglos de expresión. (Los cuales muy probablemente reflejan mejor cambios en el proteoma que aquellos inferidos mediante microarreglos donde se usa "mRNA TOTAL"..) Ergo sería interesante hacer algunos de los microarreglos que se hacen en labo usando esta técnica y ver las diferencias.
*La figura muestra en el gradiente a los mRNA sin polisomas y hasta el fondo aquellos mRNAs con múltiples polisomas.
Friday, September 25, 2009
Nucleotidos en el PCR
Nucleotides (dNTP)
dNTP "instability"
One important observation, coming from experiments with multiplex PCR, is that dNTP stocks are very sensitive to cycles of thawing/freezing. After 3-5 such cycles, multiplex PCR reactions usually did not work well. To avoid such problems, small aliquots (2-5 µl) of dNTP (25 mM each), lasting for only a couple of reactions, can be made and kept frozen at -20o C. However, during long-term freezing, small amounts of water evaporate on the walls of the vial changing the concentration of the dNTP solution. Before using, it is essential to centrifuge these vials at high speed in a microfuge.
This low stability of the dNTP is not so obvious when single loci are amplified.
Another important observation is that, anytime nucleotides are diluted in water, the solution should be buffered (for example with 10mM Tris pH 7.7-8.0, final concentration).Otherwise, an acid pH will promote hydrolysis of dNTP into dNDP and dNMP and will render them useless for enzymatic DNA polymerizing reactions.
Relationship between MgCl2 and dNTP concentration (also on page 14)
dNTP concentrations of about 200µM each are usually recommended for the Taq polymerase, at 1.5mM MgCl2 (Perkin Elmer Cetus). In a 25 µl reaction volume, theoretically these nucleotides should allow synthesis of about 6-6.5 µg of DNA. This amount should be sufficient for multiplex reactions in which 5 to 8 or more primer pairs are used at the same time. To work properly (besides the magnesium bound by the dNTP and the DNA), Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets "trapped")whereas increases in magnesium concentration often have positive effects.
The relationship between the concentration of magnesium and that of the dNTPs was investigated by performing PCR with a degenerate primer in reactions that contained 200, 400, 600 and 800 µM each dNTP, combined with 1.5, 2, 3, 4 or 5 mM MgCl2 (Fig. 34). This test confirmed that any increase in dNTP concentration requires an increase in the concentration of magnesium ions in order for the reaction to work. At 200 µM each dNTP, reaction worked at all magmesium concentrations, but for this primer it worked better at 3 mM (which is about double the recommended magnesium concentration for the amount of dNTP). At 800 µM each dNTP, reaction worked only aboove 3 mM magnesium.
(also shown on page 14)
Fig. 34. PCR with a degenerate primer at different Mg and dNTP concentrations. Each of the Mg concentrations (1.5, 2, 3, 4, 5 mM) were combined with each of the following dNTP concentrations (each): 200µM, 400µM, 600µM and 800µM. Results indicate that increasing dNTP concentrations require increasing Mg concentrations for the PCR reactions to work.
Common dNTP use in PCR and multiplex PCR
In another test aimed at examining the proper dNTP concentration, a multiplex PCR using primer mixture D was performed. The MgCl2 concentration was kept constant (3mM) while the dNTP concentration was increased stepwise from 50 to 100, 200, 400, 600 and 1200 µM each deoxynucleotide (Fig. 35). The best results were achieved at 200 and 400 µm dNTP; reaction was rapidly inhibited after these values. Lower than usual dNTP concentrations still allowed PCR amplification, but with somewhat less efficiency (lane "50").
Fig. 35. Multiplex PCR amplification of mixture D in 2x PCR buffer (3 mM Mg) using increasing concentrations of dNTP (50mM, 100mM, 200mM, 400mM, 600mM and 1200mM each). Most efficient amplification is seen at concentrations of 200-400µM each dNTP. Further increase in the dNTP concentration inhibits the reaction when MgCl2 is kept constant.
dNTP "instability"
One important observation, coming from experiments with multiplex PCR, is that dNTP stocks are very sensitive to cycles of thawing/freezing. After 3-5 such cycles, multiplex PCR reactions usually did not work well. To avoid such problems, small aliquots (2-5 µl) of dNTP (25 mM each), lasting for only a couple of reactions, can be made and kept frozen at -20o C. However, during long-term freezing, small amounts of water evaporate on the walls of the vial changing the concentration of the dNTP solution. Before using, it is essential to centrifuge these vials at high speed in a microfuge.
This low stability of the dNTP is not so obvious when single loci are amplified.
Another important observation is that, anytime nucleotides are diluted in water, the solution should be buffered (for example with 10mM Tris pH 7.7-8.0, final concentration).Otherwise, an acid pH will promote hydrolysis of dNTP into dNDP and dNMP and will render them useless for enzymatic DNA polymerizing reactions.
Relationship between MgCl2 and dNTP concentration (also on page 14)
dNTP concentrations of about 200µM each are usually recommended for the Taq polymerase, at 1.5mM MgCl2 (Perkin Elmer Cetus). In a 25 µl reaction volume, theoretically these nucleotides should allow synthesis of about 6-6.5 µg of DNA. This amount should be sufficient for multiplex reactions in which 5 to 8 or more primer pairs are used at the same time. To work properly (besides the magnesium bound by the dNTP and the DNA), Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets "trapped")whereas increases in magnesium concentration often have positive effects.
The relationship between the concentration of magnesium and that of the dNTPs was investigated by performing PCR with a degenerate primer in reactions that contained 200, 400, 600 and 800 µM each dNTP, combined with 1.5, 2, 3, 4 or 5 mM MgCl2 (Fig. 34). This test confirmed that any increase in dNTP concentration requires an increase in the concentration of magnesium ions in order for the reaction to work. At 200 µM each dNTP, reaction worked at all magmesium concentrations, but for this primer it worked better at 3 mM (which is about double the recommended magnesium concentration for the amount of dNTP). At 800 µM each dNTP, reaction worked only aboove 3 mM magnesium.
(also shown on page 14)
Fig. 34. PCR with a degenerate primer at different Mg and dNTP concentrations. Each of the Mg concentrations (1.5, 2, 3, 4, 5 mM) were combined with each of the following dNTP concentrations (each): 200µM, 400µM, 600µM and 800µM. Results indicate that increasing dNTP concentrations require increasing Mg concentrations for the PCR reactions to work.
Common dNTP use in PCR and multiplex PCR
In another test aimed at examining the proper dNTP concentration, a multiplex PCR using primer mixture D was performed. The MgCl2 concentration was kept constant (3mM) while the dNTP concentration was increased stepwise from 50 to 100, 200, 400, 600 and 1200 µM each deoxynucleotide (Fig. 35). The best results were achieved at 200 and 400 µm dNTP; reaction was rapidly inhibited after these values. Lower than usual dNTP concentrations still allowed PCR amplification, but with somewhat less efficiency (lane "50").
Fig. 35. Multiplex PCR amplification of mixture D in 2x PCR buffer (3 mM Mg) using increasing concentrations of dNTP (50mM, 100mM, 200mM, 400mM, 600mM and 1200mM each). Most efficient amplification is seen at concentrations of 200-400µM each dNTP. Further increase in the dNTP concentration inhibits the reaction when MgCl2 is kept constant.
Wednesday, August 19, 2009
Escaping fates with open states
The ability of embrionyc stem cells to give rise to any cell type relies on a remodelling protein that maintains open chromatin. But the chromatin landscape of these cells maybe more complex than previously thought.
http://www.nature.com/nature/journal/v460/n7257/pdf/nature08212.pdf
http://www.nature.com/nature/journal/v460/n7257/pdf/nature08212.pdf
Friday, August 14, 2009
Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening
Article
Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening
Piyush B. Gupta1, 3, 7, 
Summary
Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.
Tuesday, August 04, 2009
Cell death Regulation by BH3-Only family proteins.
El volumen 27, suplemento 1, de la revista Oncogene, está dedicado a la familia de proteínas con sólo el dominio BH3.
http://www.nature.com/onc/journal/v27/n1s/index.html
Monday, July 20, 2009
TD PCR
Echenle un vistazo. Nosotros usamos una modificacion de este (step down), pero pueden tratar el original
http://bitesizebio.com/2009/07/20/touchdown-pcr-a-primer-and-some-tips/
http://bitesizebio.com/2009/07/20/touchdown-pcr-a-primer-and-some-tips/
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