text:
Q: Transposing hierachical data from one VARCHAR to two INTs THE PROBLEM
The table where the data is recorded is
CREATE TABLE [dbo].[Almoxarifado](
    [idAlmoxarifado] [varchar](20) NOT NULL,
    [tipoAlmoxarifadoId] [varchar](30) NOT NULL,
    [entidadeId] [bigint] NOT NULL,
    [dtInclusao] [smalldatetime] NOT NULL,
    [dtUltimaAlteracao] [smalldatetime] NULL,
    [descricao] [varchar](255) NOT NULL,
    [terceiro] [bit] NOT NULL
) ON [PRIMARY]

And this is a SELECT TOP 10 * example (the table has more than 1 million entries):

As you can see, the idAlmoxarifadofield is storing a hierarchical data that has no hierarchical relationship as most hierarchical table have.
Now I want to bring this data to a usual hierarchical table:
CREATE TABLE [dbo].[Almoxarifado2](
    [idMaster] [bigint] IDENTITY(1,1) NOT NULL,
    [idAlmoxarifado] [int] NOT NULL,
    [idAlmoxPai] [int] NOT NULL DEFAULT ((0)),
    [entidadeId] [bigint] NOT NULL,
    [tipoAlmoxarifadoId] [varchar](30) NOT NULL
    [dtInclusao] [datetime] NULL DEFAULT (getdate()),
    [dtUltimaAlteracao] [datetime] NULL,
    [descricao] [varchar](255) NOT NULL,
    [terceiro] [bit] NULL DEFAULT ((0)),
    PRIMARY KEY ([idMaster])
)

On this table I'll have a usual father-son relationship where the "father" of a given field will always be it's idMastercorrespondent (unless it is on the first degree of the hierarchical chain; then it's "father" would be 0).
So, if we follow the same order shown on the image above, we should have (after inserting the transposed data on the new table) :

THE QUESTION
How can I do this transposition is a safe and painless way since the original table has more than 1 mi entries?
THE RULES
There are no restrictions on how many top-levels could exist; neither children levels. Our idea is to work with a mask to make the data useful on our application (we workd with Motorola Handheld Computer; check MC45 and MC3190). Let's say that the customer that buys our solution has several Warehouses where he stocks his products. In this case he'll be able to define a mask (say 9.99.99.999 or 9.9.99.99.99.999) and our system will have to be able to use this mask to allow him to register his warehouses in those levels.
So you can basically say that our system is foccused on logistics in a fashion.
Where the 1st level could be "NY South Warehouse", the 2nd "Room #2", 3rd "Corridor A", 4th "Locker 2" and 5th "Shelf 3".

A: Add a column to dbo.Almoxarifado2 that will hold the original value of idAlmoxarifado.
Add all rows from dbo.Almoxarifado and then use idAlmoxarifadoOrg to update idAlmoxPai with the value of idMaster for the parent row.
Code with some comments:
-- Add a column to store original idAlmoxarifado
alter table dbo.Almoxarifado2
    add idAlmoxarifadoOrg varchar(20) not null;

go

-- Add all rows to Almoxarifado2 with 0 to idAlmoxPai
-- and the rightmost number of idAlmoxarifado to idAlmoxarifado.
-- Original value of idAlmoxarifado goes to idAlmoxarifadoOrg
insert into dbo.Almoxarifado2(idAlmoxarifado, idAlmoxPai, entidadeId, tipoAlmoxarifadoId, dtInclusao, dtUltimaAlteracao, descricao, terceiro, idAlmoxarifadoOrg)
select right(A.idAlmoxarifado, charindex('.', reverse('.'+A.idAlmoxarifado)) - 1), 0, A.entidadeId, A.tipoAlmoxarifadoId, A.dtInclusao, A.dtUltimaAlteracao, A.descricao, A.terceiro, A.idAlmoxarifado
from dbo.Almoxarifado as A;

go

-- Update idAlmoxPai with the parent idMaster
-- Don't update root nodes
update A
set idAlmoxPai = P.idMaster
from dbo.Almoxarifado2 as A
  inner join dbo.Almoxarifado2 as P
    on P.idAlmoxarifadoOrg = left(A.idAlmoxarifadoOrg, len(A.idAlmoxarifadoOrg) - charindex('.', reverse(A.idAlmoxarifadoOrg)))
where charindex('.', A.idAlmoxarifadoOrg) > 0;

go

-- Cleanup
alter table dbo.Almoxarifado2 drop column idAlmoxarifadoOrg;

Some parts could do with some more information.
This right(A.idAlmoxarifado, charindex('.', reverse('.'+A.idAlmoxarifado)) - 1) extracts the last number in idAlmoxarifado. So 1.1.10 will give you a 10.
And this part left(A.idAlmoxarifado, len(A.idAlmoxarifado) - charindex('.', reverse(A.idAlmoxarifado))) returns all but the last number. For 1.1.10 it will return 1.1.
I shamelessly stole the test data generated by wBob (thanks and much appreciated) and found out that this version is faster on my machine. I use a really ancient 2 core laptop for the tests so the result could be different on a real server. Any way, for me it took 3 minutes to execute the code by wBob and my solution was about 20 seconds.

A: As your data looks a bit like hierarchyId I thought about using that.  Initial versions did not scale well up to 1 million rows, however a few indexing choices on the main temp table have helped.  However issues could also be due to my test data, so can you tell me a bit more about your hierarchy?  For example how many top-level parents are there, how many levels are there on average, how many children might each level have?
In the meantime, here is a version so you can have a look through it and see if hierarchyId could work for you.
The basic idea is to create a temp table with the original ids converted into hierarchyIds, then walk the hierarchy to work out the lineage.  This temp table (which could be permanent) could then be used for the migration:
USE tempdb
GO

SET NOCOUNT ON
GO

IF OBJECT_ID('[dbo].[Almoxarifado]') IS NOT NULL DROP TABLE [dbo].[Almoxarifado]
IF OBJECT_ID('[dbo].[Almoxarifado2]') IS NOT NULL DROP TABLE [dbo].[Almoxarifado2]
GO

CREATE TABLE [dbo].[Almoxarifado](
    [idAlmoxarifado] [varchar](20) NOT NULL,
    [tipoAlmoxarifadoId] [varchar](30) NOT NULL,
    [entidadeId] [bigint] NOT NULL,
    [dtInclusao] [smalldatetime] NOT NULL,
    [dtUltimaAlteracao] [smalldatetime] NULL,
    [descricao] [varchar](255) NOT NULL,
    [terceiro] [bit] NOT NULL
) ON [PRIMARY]
GO

CREATE TABLE [dbo].[Almoxarifado2](
    [idMaster] [bigint] IDENTITY(1,1) NOT NULL,
    [idAlmoxarifado] [int] NOT NULL,
    [idAlmoxPai] [int] NOT NULL DEFAULT ((0)),
    [entidadeId] [bigint] NOT NULL,
    [tipoAlmoxarifadoId] [varchar](30) NOT NULL ,
    [dtInclusao] [datetime] NULL DEFAULT (getdate()),
    [dtUltimaAlteracao] [datetime] NULL,
    [descricao] [varchar](255) NOT NULL,
    [terceiro] [bit] NULL DEFAULT ((0)),
    PRIMARY KEY ([idMaster])
)
GO

/*
-- Test data
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
VALUES
    ( '1', 'TESTE', 12, '29 Apr 2016', NULL, '0000000', 0 ),
    ( '1.1', 'TESTE', 12, '29 Apr 2016', NULL, '0000001', 0 ),
    ( '1.1.01', 'TESTE', 12, '29 Apr 2016', NULL, '0000002', 0 ),
    ( '1.1.01.01', 'TESTE', 12, '29 Apr 2016', NULL, '0000003', 0 ),
    ( '1.1.01.01.001', 'TESTE', 12, '29 Apr 2016', NULL, '0000004', 0 ),

    ( '1.1.01.01.002', 'TESTE', 12, '29 Apr 2016', NULL, '0000005', 0 ),
    ( '1.1.01.01.003', 'TESTE', 12, '29 Apr 2016', NULL, '0000006', 0 ),
    ( '1.1.01.01.004', 'TESTE', 12, '29 Apr 2016', NULL, '0000007', 0 ),
    ( '1.1.01.01.005', 'TESTE', 12, '29 Apr 2016', NULL, '0000008', 0 ),
    ( '1.1.01.01.006', 'TESTE', 12, '29 Apr 2016', NULL, '0000009', 0 )
GO
*/

-- Add 10 parent levels
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
VALUES
    ( '1', 'TESTE', 1, '1 Jan 2016', NULL, '00000001', 0 ),
    ( '2', 'TESTE', 1, '1 Feb 2016', NULL, '00000002', 0 ),
    ( '3', 'TESTE', 1, '1 Mar 2016', NULL, '00000003', 0 ),
    ( '4', 'TESTE', 1, '1 Apr 2016', NULL, '00000004', 0 ),
    ( '5', 'TESTE', 1, '1 May 2016', NULL, '00000005', 0 ),
    ( '6', 'TESTE', 1, '1 Jun 2016', NULL, '00000006', 0 ),
    ( '7', 'TESTE', 1, '1 Jul 2016', NULL, '00000007', 0 ),
    ( '8', 'TESTE', 1, '1 Aug 2016', NULL, '00000008', 0 ),
    ( '9', 'TESTE', 1, '1 Sep 2016', NULL, '00000008', 0 ),
    ( '10', 'TESTE', 1, '1 Oct 2016', NULL, '00000010', 0 )

-- For each parent, add 3 sublevels
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
SELECT
    idAlmoxarifado + '.' + CAST( x.y AS VARCHAR(10) ),
    tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro
FROM [dbo].[Almoxarifado] a
    CROSS JOIN ( SELECT TOP 5 idAlmoxarifado y FROM [dbo].[Almoxarifado] ) x

-- add n sublevels
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
SELECT
    idAlmoxarifado + '.0' + CAST( x.y AS VARCHAR(10) ),
    tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro
FROM [dbo].[Almoxarifado] a
    CROSS JOIN ( SELECT TOP 8 idAlmoxarifado y FROM [dbo].[Almoxarifado] WHERE idAlmoxarifado Not Like '%.%' ) x
WHERE idAlmoxarifado Like '%.%'

-- Add 8 sublevels
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
SELECT
    idAlmoxarifado + '.0' + CAST( x.y AS VARCHAR(10) ),
    tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro
FROM [dbo].[Almoxarifado] a
    CROSS JOIN ( SELECT TOP 13 ROW_NUMBER() OVER( ORDER BY idAlmoxarifado ) y FROM [dbo].[Almoxarifado] ) x
WHERE idAlmoxarifado Like '%.%.%'

-- Add 9 sublevels
INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
SELECT
    idAlmoxarifado + '.0' + CAST( x.y AS VARCHAR(10) ),
    tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro
FROM [dbo].[Almoxarifado] a
    CROSS JOIN ( SELECT TOP 21 ROW_NUMBER() OVER( ORDER BY idAlmoxarifado ) y FROM [dbo].[Almoxarifado] ) x
WHERE idAlmoxarifado Like '%.%.%.%'
GO

INSERT INTO [dbo].[Almoxarifado] ( idAlmoxarifado, tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro )
SELECT
    idAlmoxarifado + '.0' + CAST( x.y AS VARCHAR(10) ),
    tipoAlmoxarifadoId, entidadeId, dtInclusao, dtUltimaAlteracao, descricao, terceiro
FROM [dbo].[Almoxarifado] a
    CROSS JOIN ( SELECT TOP 9 ROW_NUMBER() OVER( ORDER BY idAlmoxarifado ) y FROM [dbo].[Almoxarifado] ) x
WHERE idAlmoxarifado Like '%.%.%.%.%'
GO

-- Main hierarchyId processing
IF OBJECT_ID('tempdb..#tmp') IS NOT NULL DROP TABLE #tmp

SELECT
    IDENTITY( INT, 1, 1 ) AS idMaster,
    idAlmoxarifado,
    CAST( '/' + REPLACE( REPLACE( REPLACE( idAlmoxarifado, '.0', '.' ), '.0', '.' ), '.', '/' ) + '/' AS hierarchyid ) hId,
    CAST( '/' + REPLACE( REPLACE( REPLACE( idAlmoxarifado, '.0', '.' ), '.0', '.' ), '.', '/' ) + '/' AS hierarchyid ).ToString() hString,
    CAST( NULL AS INT ) AS idAlmoxarifado2,
    CAST( NULL AS INT ) AS idAlmoxPai

INTO #tmp
FROM [dbo].[Almoxarifado]
GO

-- Index temp table to help with recursive CTE
ALTER TABLE #tmp ADD PRIMARY KEY ( idMaster )
ALTER TABLE #tmp ALTER COLUMN hId hierarchyId NOT NULL
CREATE UNIQUE INDEX _idx ON #tmp ( hId ) INCLUDE ( idAlmoxarifado, hString )
GO

-- Walk the hierarchy
;WITH cte AS (
SELECT 1 AS xlevel, idMaster, idAlmoxarifado, hId, hString, hId.GetLevel() getLevel, 0 AS parentId
FROM #tmp
WHERE hId.GetLevel() = 1

UNION ALL

SELECT xlevel + 1, t.idMaster, t.idAlmoxarifado, t.hId, t.hString, t.hId.GetLevel() getLevel, c.idMaster AS parentId
FROM cte c
    INNER JOIN #tmp t ON c.hId  = t.hId.GetAncestor(1)
)
UPDATE t
SET
    -- Parse out the individual node, by taking its parent and stuffing it in front
    t.idAlmoxarifado2 = CAST( REPLACE( STUFF( c.hString, 1, LEN(c.hId.GetAncestor(1).ToString()), '' ), '/', '' ) AS INT ),
    t.idAlmoxPai = parentId
FROM cte c
    INNER JOIN #tmp t ON c.idMaster = T.idMaster

-- Check results
SELECT *
FROM #tmp

This whole script (including test data creation) runs in only a few minutes on my laptop.

redpajama_set_name: RedPajamaStackExchange


text:
Focus on Radiation Oncology
Novel Combinations
Valproic acid, in combination with all-trans retinoic acid and 5-aza-2′-deoxycytidine, restores expression of silenced RARβ2 in breast cancer cells
Nigel P. Mongan and Lorraine J. Gudas
Nigel P. Mongan
Lorraine J. Gudas
DOI: 10.1158/1535-7163.MCT-04-0079 Published March 2005
Epigenetic silencing of tumor suppressor genes has been established as an important process of carcinogenesis. The retinoic acid (RA) receptor β2 (RARβ2) gene is one such tumor suppressor gene often silenced during carcinogenesis. The combined use of histone deacetylase and DNA methyltransferase inhibitors has been shown to reverse the epigenetic silencing of numerous growth regulatory genes. Valproic acid (VPA), which has long been used in the treatment of epilepsy, was shown recently to be an effective histone deacetylase inhibitor that can induce differentiation of neoplastically transformed cells. In this study, we show for the first time that VPA, in combination with RA and the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC), can overcome the epigenetic barriers to transcription of a prototypical silenced tumor suppressor gene, RARβ2, in human breast cancer cells. Chromatin immunoprecipitation assays show that the combination of VPA, RA, and Aza-dC increases histone acetylation at the silenced RARβ2 promoter of MCF-7 breast cancer cells. Furthermore, reverse transcription-PCR analyses reveal cell type–specific effects in the actions of VPA on RARβ2 expression in cultured human breast cancer cells. Finally, we show that VPA, in combination with RA and Aza-dC, inhibits the proliferation of both estrogen receptor α-positive (MCF-7) and estrogen receptor α-negative (MDA-MB-231) breast cancer cell lines. These data suggest that VPA may ultimately be useful in combination therapies in the treatment of human breast cancers.
5-aza-2′-deoxycytidine
HDAC inhibitor
retinoic acid receptor
tumor suppressor
Histone deacetylases (HDAC; ref. 1) and DNA methyltransferases (DNMT; ref. 2) play essential roles in the developmental and temporal regulation of gene expression. However, epigenetic silencing of tumor suppressor genes has emerged as an important process of carcinogenesis (3). It has been shown that HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and the butyrates, can induce differentiation and inhibit proliferation of many cancer cell types in vitro (4). The anticancer actions of HDAC inhibitors are attributable at least in part to the restoration and up-regulation of multiple functionally distinct growth inhibitory pathways (5–8). Indeed, the use of HDAC and DNMT inhibitors has been shown to restore the expression of silenced estrogen receptor α (ERα; ref. 9), retinoic acid (RA) receptor β (RARβ; refs. 10–12), and numerous other growth regulatory genes (5, 6, 13, 14). For these reasons, HDAC and DNMT inhibitors have emerged as promising candidates for differentiation therapy of many cancer types (4, 15–18).
RA, the primary physiologic metabolite of vitamin A, plays essential roles in normal development, growth, and cellular differentiation (19). Clinical trials are now under way to investigate the use of HDAC inhibitors, alone and in combination with RA and/or DNMT inhibitors, for the treatment of solid tumors (4), as both RA (20, 21) and HDAC inhibitors have shown promising results for the treatment of acute promyelocytic leukemia (17). The signaling actions of RA are mediated by RA receptors, RARs and retinoid X receptors, which are members of the nuclear receptor superfamily of ligand-dependent transcription factors (19). There is evidence that RARα (14), RARβ2 (22–25), and RARγ (26) can each act as tumor suppressors, and their expression is reduced in breast cancer cells (27). However, the antineoplastic pathways induced by RA are regulated predominantly by RARβ2 (24, 25). However, expression of RARβ2 is frequently decreased or lost in many cancer types (28–30), including breast cancer (12, 23, 27, 31). The RARβ2 P2 promoter possesses a RA response element (RARE) normally transactivated by a retinoid X receptor-RAR heterodimer in the presence of RA (32). Recent studies have shown that the RARβ2-RARE is epigenetically silenced in breast (10–12) and other cancer types (28, 30, 33). This combination of local histone deacetylation and CpG island methylation results in strong epigenetic repression of RARβ2 and resistance to the growth inhibitory effects of RA (27). However, the combined use of RA with HDAC and/or DNMT inhibitors has shown synergistic effects in restoring the transcription and growth regulatory effects of silenced RARβ2 (10, 11, 23). Furthermore, restoration of RARβ2 expression in MCF-7 (23) and MDA-MB-231 (34) breast cancer cells has been shown to restore the growth inhibitory and proapoptotic actions of RA.
Valproic acid (2-propylpentanoic acid; VPA) is a short-chain fatty acid that is structurally related to the butyrate class of HDAC inhibitors (4) and was shown recently to be an effective HDAC inhibitor at concentrations currently in use for the treatment of epilepsy (35–37). VPA is also known to stimulate differentiation and/or inhibit cell growth of cultured leukemic (8, 37), endometrial (38), and glioma cells (39) and diminishes adenoma formation in the APCmin colon cancer mouse model (40). VPA is a particularly attractive candidate HDAC inhibitor for use in the treatment of neoplastic disease, as it is generally well tolerated by patients (41). Although clinical trials with VPA for cancer are under way (4, 42), the effects of VPA on the proliferation and tumor suppressor gene expression have not yet been reported for breast cancer cells. In this study, we have investigated the HDAC inhibitory activity of VPA in the context of a prototypical epigenetically silenced tumor suppressor gene, RARβ2, in human breast cancer cells. Of particular significance to future mouse xenograft and clinical studies, we investigated the efficacy of combinations of low concentrations of VPA with RA and/or the DNMT inhibitor 5-aza-2′-deoxycytidine (Aza-dC/Decitabine), which has been shown in previous studies to enhance reactivation of silenced RARβ2 in cancer cells (10, 23, 28, 43, 44). We show that the combination of VPA, RA, and Aza-dC increases histone H3 acetylation at the silenced RARβ2-RARE in MCF-7 breast cancer cells. Consistent with this, expression of RARβ2 is restored or enhanced in several human breast cancer cell types treated with VPA, RA, and Aza-dC. Furthermore, we show that VPA, in combination with RA and Aza-dC, inhibits proliferation of both ERα-positive and ERα-negative breast cancer cell lines. These data suggest that VPA may be useful in the treatment of breast cancer.
Cell Culture, Drug Treatments, and Growth Analyses
Human breast cancer cell lines MCF-7, MDA-MB-231, SK-BR3, MDA-MB-453, and HS578T were maintained as directed by the American Type Culture Collection (Manassas, VA). The MDA-MB-231 clone employed in this study expresses RARβ2 following RA treatment as reported previously (11, 45). Cells were placed in six-well plates at a density of 1 × 105 cells per well 24 hours before drug treatment to allow cell attachment. All-trans RA (Sigma, St. Louis, MO) was dissolved in ethanol and used at a final concentration of 1 μmol/L. RA is essential to induce expression of RARβ2 via the RARE (32). Aza-dC (Sigma) was dissolved in PBS (pH 6.8) and used at a final concentration of 2 μmol/L, a concentration within the range employed previously (1-10 μmol/L; refs. 28, 43). VPA was purchased from Sigma and dissolved in sterile water and used at a final concentration of 250 μmol/L or as indicated in the figure legend (Fig. 5). Cells were treated with fresh medium and drug combinations twice (at 0 and 48 hours) during a 96-hour period. Under these conditions, dividing cells remain subconfluent, thereby permitting incorporation of Aza-dC throughout the treatment time course. Control cells were treated with ethanol. For growth analyses, cells were counted every 24 hours using a Coulter counter (Fullerton, CA). Percentage growth inhibition was determined as TC/CC × 100, where TC represents treated cells and CC represents control cell numbers. The growth experiments were done at least four times.
Epigenetic Status of the RARβ2-RARE P2
Chromatin immunoprecipitation (ChIP) assays were done as described previously (23, 46) using ChIP-grade antibodies to acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY). Cells were placed in six-well plates at a density of 1 × 105 cells per well 24 hours before drug treatment to allow cell attachment. Chromatin was isolated from cells treated twice with fresh medium and drug combinations (at 0 and 48 hours) during a 96-hour period. At the conclusion of the drug treatment, RA (1 μmol/L) was added to all cells for 30 minutes immediately before the ChIP assay. This ensures that all cells are exposed to equivalent concentrations of RA for the ChIP assays and compensates for defects in retinoid metabolism, which render MCF-7 cells incapable of accumulating retinoids (47, 48). During this 30-minute period, β-estradiol (100 nmol/L) was also added to the cells to enhance transcription at the PS2 estrogen response element and acts as a positive control for nuclear receptor–mediated transcription. Cells were then cross-linked with formaldehyde (1%) for 10 minutes at 37°C. Cross-linked samples were sonicated thrice for 10 seconds each at output level 3 using a Branson sonifier model 150, to yield an average DNA size of 200 to 800 bp, as confirmed by electrophoresis on 2% TAE-agarose gels. Soluble chromatin was immunoprecipitated overnight with antibodies specific for acetylated histone H3. The immunoprecipitated chromatin complex was harvested using protein A-agarose beads (Upstate Biotechnology). DNA was extracted using the QIAquick extraction system (Qiagen, Valencia, CA) and eluted in a volume of 50 μL ultrapure water. PCR was carried out as described below, with the number of cycles determined by that required to yield amplification within the linear range. Primers specific for the RARβ2-RARE P2 promoter (192-bp product) were forward 5′-CTCTGGCTGTCTGCTTTTGC-3′ and reverse 5′-CAGCTCACTTCCTACTACTTC-3′. Primers specific for the estrogen-responsive ER response element of the PS2 promoter (323-bp product; ref. 46) and GAPDH (265-bp product; ref. 23) were used as internal positive controls as described previously. Negative control PCRs using water in place of template were done for each experiment.
RNA Isolation, Reverse Transcription-PCR, and Real-time PCR Quantification
Total RNA was isolated from cells using Trizol (Invitrogen, Carlsbad, CA) and first-strand cDNA synthesized from 2 to 5 μg total RNA using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) as directed by the manufacturer. The synthesized cDNA was diluted to 100 μL with ultrapure water. Primers specific for the β-actin cDNA (Genbank accession no. NM001101) were used to confirm cDNA integrity and oligonucleotide primers were designed to amplify the RARα (accession no. BC008727), RARβ2 (accession nos. M96016-M96023), and RARγ (M24857) cDNAs. Primer pairs used in reverse transcription-PCR (RT-PCR) were as shown in Table 1. Primers were designed to span a least one intron-exon boundary and thereby prevent amplification of any contaminating genomic DNA. The RT-PCR products of each primer pair were sequenced to confirm the identity of the amplified fragment and the specificity of amplification. Each PCR contained 4 ng of each oligonucleotide, 1 μL cDNA, 0.5 unit Taq DNA polymerase and accompanying 1× buffer (Invitrogen), 1.5 mmol/L MgCl2, and 0.2 mmol/L deoxynucleotide triphosphates. Thermal cycling was done as follows: 95°C for 5 minutes followed by 33 to 40 cycles of 94°C for 30 seconds (template denaturation), 55°C for 30 seconds (oligonucleotide annealing), and 72°C for 30 seconds (product extension). PCR products were analyzed by electrophoresis through 1.5% TAE-agarose gels.
Sequences of primer pairs used to assess mRNA expression using RT-PCR
Real-time PCR analysis was done using a DNA Engine Opticon System (MJ Research, Boston, MA) and SYBR Green I Quantitect kit (Qiagen). The real-time PCR technique employed enables the detection and quantification of very low levels of RARβ2 expression in breast cancer cells not possible with end point RT-PCR. The threshold cycle (CT) was set at 10 times the SD above the mean baseline emission for cycles 3 to 7. Expression of β-actin was unaffected by drug treatments and therefore used as an internal reference. Primer pairs for RARβ2 and β-actin were as described above. Amplification efficiencies of these primer pairs were compared using template dilutions and found to have similar ΔCT values (ΔCT = CT RARβ2 − CT β-actin). Reaction conditions were 95°C for 10 minutes to activate the polymerase followed by 50 cycles of 94°C for 30 seconds, primer annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; fluorescence was read after each cycle at 82°C. The relative levels of RARβ2 expression for each cell type and treatment condition were determined with reference to the internal β-actin control using the 2−ΔΔCT method (49, 50). With the exception of MDA-MB-453, expression of RARβ2 was detected in all cell lines treated with RA; hence, this sample was chosen as the calibrator sample with an arbitrary normalized value of 1. In MDA-MB-453, the calibrator sample was derived from RA- and Aza-dC-treated cells. Real-time analysis was done in triplicate for each cDNA sample. Negative control PCRs using water in place of template were done for each experiment.
VPA, in Combination with RA and Aza-dC, Restores Expression of RARβ2 in MCF-7 Breast Cancer Cells
RT-PCR was used to monitor RARβ2 expression in MCF-7 breast cancer cells. Expression of RARβ2 was not detected in untreated control cells (Fig. 1A, lane 1) and not induced in MCF-7 cells following treatment with RA (1 μmol/L) alone or in combination with VPA (250 μmol/L) or Aza-dC (2 μmol/L) for 96 hours (Fig. 1A, lanes 2-6). Indeed, expression of RARβ2 mRNA was detected only in MCF-7 cells treated simultaneously with RA, Aza-dC, and VPA (Fig. 1A, lane 7).
A, cells were treated twice over a 96-h period with ethanol (lane 1), RA (1 μmol/L; lane 2), VPA (250 μmol/L; lane 3), RA + VPA (lane 4), Aza-dC (2 μmol/L; lane 5), RA + Aza-dC (lane 6), and RA + Aza-dC + VPA (lane 7) and RT-PCR was done as described in Materials and Methods. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gel and stained with ethidium bromide. B and C, ChIP assays using an antibody to acetylated histone H3 were used to assess the ability of 1 μmol/L RA (B, lane 1) or 2 μmol/L Aza-dC (B, lane 2) in combination with differing VPA concentrations (C, lanes 1-5) to restore histone H3 acetylation to the RARβ2 P2 promoter. Cells were treated twice over a 96-h period with RA and Aza-dC in combination with 5 mmol/L VPA (C, lane 1), 3 mmol/L VPA (C, lane 2), 1 mmol/L VPA (C, lane 3), 500 μmol/L VPA (C, lane 4), 250 μmol/L VPA (C, lane 5), RA + Aza-dC (C, lane 6), and vehicle control (C, lane 7). Immediately before the ChIP assay, RA (1 μmol/L) and β-estradiol (100 nmol/L) were added to the cells for 30 minutes before formaldehyde cross-linking and histone H3 acetylation assessed at the RARβ2-RARE. The promoter regions of PS2 and GAPDH act as internal positive controls. Thirty-two (B) and 33 (C) cycles of PCR were used in the ChIP experiments. PCRs were analyzed by electrophoresis on a 2% TAE-agarose gel and repeated yielding similar results. βE2, β-estradiol; C, Aza-dC; IP, immunoprecipitation with anti–histone H3 antibody; R, RA; V, VPA.
Histone H3 deacetylation is believed to be an important marker of the epigenetic silencing of the RARβ2-RARE promoter (28). ChIP assays were therefore employed to examine the effects of VPA in combination with RA and Aza-dC on histone H3 acetylation associated with the RARβ2-RARE of MCF-7 breast cancer cells. Consistent with the RT-PCR analysis (Fig. 1A), treatment of MCF-7 cells with RA or Aza-dC alone failed to increase histone H3 acetylation at the RARβ2-RARE (Fig. 1B). This absence of histone H3 acetylation in MCF-7 cells following RA treatment provided further evidence of the epigenetic silencing of the RARβ2-RARE P2 in MCF-7 cells (10). The estrogen-responsive PS2-ER response element acted as a positive control for nuclear receptor–stimulated histone H3 acetylation and GAPDH acted as a control for constitutively active chromatin (Fig. 1B). As expression of RARβ2 was detected only in MCF-7 cells treated simultaneously with VPA, RA, and Aza-dC (Fig. 1A), we next examined the effects of RA and Aza-dC in combination with increasing VPA concentrations (Fig. 1C) on RARβ2-RARE-associated histone H3 acetylation. VPA concentrations examined ranged from 5 mmol/L VPA, which was examined in early biochemical (35) and cellular (38) studies, to 250 μmol/L VPA, a concentration of more pharmacologic relevance (51, 52). At the conclusion of the 96-hour drug treatment, RA (1 μmol/L) and β-estradiol (100 nmol/L) were added to the cells for 30 minutes and the effects on histone H3 acetylation at the RARβ2-RARE and PS2-ER response element were examined using ChIP assays. The estrogen-responsive PS2-ER response element and GAPDH acted as a positive controls for active chromatin. Whereas all concentrations of VPA, in combination with RA and Aza-dC, could increase histone H3 acetylation associated with the RARβ2-RARE in MCF-7 cells, lower VPA concentrations (250 μmol/L-3 mmol/L) were more effective in inducing histone acetylation at the MCF-7 RARβ2-RARE than the drug combination employing 5 mmol/L VPA (Fig. 1C). It is possible that 5 mmol/L VPA may directly inhibit proliferation of MCF-7 cells and this is reflected in decreased H3 acetylation (Fig. 1C, lane 1) relative to that detected in cells treated with lower VPA concentrations (Fig. 1C, lanes 2-5). Indeed, VPA is known to induce apoptosis of cultured endometrial cancer cells in a dose-dependent manner (38). For these reasons, this lower VPA concentration (250 μmol/L), which is within the range of mean serum levels (260-500 μmol/L), achieved in epilepsy patients treated with VPA (51, 52), was employed in RT-PCR experiments (Figs. 1A, 2, and 3) and growth analyses (Fig. 4). Although previous studies have investigated the antitumor effects of VPA in neuroblastoma (53) and endometrial mouse xenograft (38) studies, these data suggest that future breast cancer xenograft studies may wish to examine VPA alone and in combination with RA and DNMT inhibitors.
RT-PCR was used to detect expression of RARα (314 bp), RARβ (256 bp), and RARγ (394 bp) in MCF-7 (A), MDA-MB-231 (B), MDA-MB-453 (C), SK-BR3 (D), and HS578T (E) breast cancer cells. Amplification of β-actin (379 bp) was used to confirm cDNA integrity. Cells were treated twice over a 96-h period with ethanol (lane 1), RA (1 μmol/L; lane 2), VPA (250 μmol/L; lane 3), RA + VPA (lane 4), Aza-dC (2 μmol/L; lane 5), RA + Aza-dC (lane 6), and RA + Aza-dC + VPA (lane 7) and RT-PCR was done as described in Materials and Methods. A nonspecific amplification product was observed in control and VPA-treated MDA-MB-231 cells (B) and control MDA-MB-453 cells (C). The identity of the PCR product of each primer pair was confirmed by automated DNA sequencing. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gel and stained with ethidium bromide.
Real-time PCR (A-D) was done as described in Materials and Methods. Cells were treated twice over a 96-h period with RA (1 μmol/L), VPA (250 μmol/L), RA + VPA, Aza-dC (2 μmol/L), RA + Aza-dC, and RA + Aza-dC + VPA. The expression of RARβ2 was measured relative to cellular β-actin levels and quantified relative to calibrator sample (asterisks). Real-time PCR analysis was done in triplicate for each sample. Columns, mean; bars, SD. No signal indicates that RARβ2 was not detected under the conditions used. R, RA (1 μmol/L); V, VPA (250 μmol/L); C, Aza-dC (2 μmol/L); Con, control.
Proliferation of MCF-7 (A-C) and MDA-MB-231 (D-F) breast cancer cells was monitored in cells treated twice with combinations of VPA (250 μmol/L), RA (1 μmol/L), and Aza-dC (2 μmol/L) during the 96-h growth experiment. Growth inhibition expressed as percentage of untreated control MCF-7 (C) and MDA-MB-231 (F) cells is displayed to permit comparison between experiments done on different occasions. Growth assays were done on a minimum of four occasions. Points, mean; bars, SE.
VPA, RA, and Aza-dC Restore and Enhance RARβ2 Expression in Breast Cancer Cells
RT-PCR was used to detect retinoid receptor transcripts in several breast cancer cell types treated with VPA (250 μmol/L), RA (1 μmol/L), and Aza-dC (2 μmol/L; Fig. 2). Constitutive expression of RARα and RARγ was detected in all breast cancer cell lines examined using RT-PCR (Fig. 2). However, cell type–specific differences in RARβ2 expression were observed. As described above, expression of RARβ2 was only detected in MCF-7 cells following treatment with RA, VPA, and Aza-dC (Fig. 1A). In MDA-MB-231, RARβ2 expression was detected in cells treated with RA alone (11) and in combination with either VPA and/or Aza-dC (Fig. 2B). Expression of RARβ2 mRNA was detected in MDA-MB-453 cells treated with RA and Aza-dC or treated simultaneously with all three drugs (Fig. 2C). As in MDA-MB-231, RARβ2 expression was detected in SK-BR3 cells treated with RA alone and in combination with VPA and Aza-dC (Fig. 2D). Constitutive RARβ2 expression was detected in HS578T breast cancer cells (Fig. 2E) as reported previously (10, 27).
Real-time PCR was used to quantify changes in RARβ2 expression in breast cancer cells induced at 96 hours following drug treatments. This approach enabled the relative quantification of low levels of RARβ2 expression in breast cancer cells not possible with end point RT-PCR (Fig. 2). The effect of VPA alone and in combination with Aza-dC on RA-induced expression of RARβ2 was compared with the basal levels of RARβ2 detected in each breast cancer cell line (Fig. 3). In MCF-7, similar levels of RARβ2 expression were found in the presence of RA, VPA, or Aza-dC (Fig. 3A), suggesting that this represents a background level of expression from a basal promoter (23). The combined use of RA, VPA, and Aza-dC resulted in ∼14-fold increase in RARβ2 mRNA expression relative to RA-treated MCF-7 cells. In MDA-MB-231 cells, RA, in combination with VPA or Aza-dC or both, resulted in a similar (6-fold) increase in RARβ2 expression relative to RA treatment alone (Fig. 3B). Expression of RARβ2 was detected only in MDA-MB-453 cells treated simultaneously with RA and Aza-dC. However, the level of RARβ2 mRNA was increased in MDA-MB-453 cells when VPA was used in addition to RA and Aza-dC (Fig. 3C). Combined treatment of SK-BR3 cells with RA, VPA, and Aza-dC resulted in the greatest enhancement of RARβ2 expression relative to the RA-treated control (Fig. 3D). Although in all breast cancer cell lines tested the combined use of VPA, RA, and Aza-dC resulted in maximal reactivation of RARβ2 expression, the different levels of RARβ2 detected in each cell line treated with each combination of RA, VPA, and Aza-dC suggest that distinct repressive states may exist at the RARβ2-RARE.
VPA, Alone and in Combination with RA and Aza-dC, Inhibits Breast Cancer Cell Proliferation
We next examined the effects of VPA, alone and with RA and Aza-dC, on proliferation of MCF-7 (Fig. 4A-C) and MDA-MB-231 (Fig. 4D-F). Cells were treated twice during a 96-hour time course, with VPA (250 μmol/L), RA (1 μmol/L), and Aza-dC (2 μmol/L). Consistent with previous reports (27), MCF-7 cells were partially sensitive, whereas MDA-MB-231 cells were resistant to RA-induced growth inhibition. In MCF-7 cells, growth inhibition (Fig. 4C) coincided with restoration of RARβ2 expression (Fig. 3A). However, growth inhibition (Fig. 4C) was also observed in MCF-7 cells treated with drug combinations that failed to increase RARβ2 levels at 96 hours (Fig. 3A). Similarly, MDA-MB-231 cells were growth inhibited by Aza-dC treatment alone, which failed to induce expression of RARβ2. Growth inhibition was also observed in MDA-MB-231 cells treated with RA with Aza-dC and/or VPA (Fig. 4), which increased RARβ2 expression. Yet, enhancement of RARβ2 expression in MDA-MB-231 cells (Fig. 3B) by treatment with RA and VPA did not coincide with growth inhibition (Fig. 4D). Similar growth inhibition was achieved in breast cancer cells treated with RA, Aza-dC, and varying VPA (250-50 μmol/L) concentrations (Fig. 5). Hence, in MCF-7, growth inhibition could occur in the absence of detectable RARβ2 expression, whereas in MDA-MB-231 restoration of RARβ2 by RA and VPA failed to inhibit proliferation. These results indicate that additional tumor suppressors, in addition to RARβ2, play essential roles in inducing growth inhibition following treatment with RA, HDAC, and DNMT inhibitors.
RT-PCR was used to detect expression of RARβ in MCF-7 (A), MDA-MB-231 (C), MDA-MB-453 (E), and SK-BR3 (G) breast cancer cells. Amplification of β-actin was used to confirm cDNA integrity. Cells were treated twice over a 96-h period with ethanol, RA (1 μmol/L), and Aza-dC (2 μmol/L) with decreasing VPA concentrations (lane 1); RA + Aza-dC + VPA (250 μmol/L; lane 2); RA + Aza-dC + VPA (100 μmol/L; lane 3); RA + Aza-dC + VPA (50 μmol/L; lane 4); and RA + Aza-dC (lane 5). RT-PCR was done as described in Materials and Methods. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gels and stained with ethidium bromide. Proliferation of MCF-7 (B), MDA-MB-231 (D), MDA-MB-453 (F), and SK-BR3 (H) breast cancer cells was monitored in cells treated twice with combinations of RA (1 μmol/L) with Aza-dC (2 μmol/L) and varying VPA concentrations as indicated during the 96-h growth experiment. Cell number was measured after 96-h growth. Growth assays were done in quadruplicate. Columns, mean; bars, SE.
The use of chromatin-modifying drugs to restore or enhance endogenous antiproliferative mechanisms is an attractive therapeutic approach for cancer (16). Indeed, HDAC inhibitors have emerged as promising candidates for differentiation therapy of both hematologic and solid tumors (4, 17, 18). Although HDAC inhibitors are believed to influence expression of <2% to 10% of genes (37, 54), HDAC inhibitors can contribute to the reactivation of multiple genes silenced during carcinogenesis and thereby restore function of key regulatory pathways, which can inhibit of cancer cell proliferation (55). Previous studies have shown the efficacy of structurally distinct HDAC and DNMT inhibitors in inducing differentiation, growth arrest, and apoptosis of some cancer cell types (16–18). Several HDAC inhibitors are undergoing clinical trials (4); however, some may be of less therapeutic utility due to rapid metabolism and/or presence of multiple nonselective actions (ref. 37 and references therein).
In this context, VPA is an attractive candidate for cancer differentiation therapy. Whereas the VPA concentration required to induce histone acetylation in vivo is not currently known, VPA is an effective HDAC inhibitor in vitro at concentrations commonly used in the treatment of epilepsy. Furthermore, the VPA concentration used in this study (250 μmol/L) is within the range of mean serum levels (260-500 μmol/L) achieved in epilepsy patients treated with VPA (51, 52). Although VPA is largely bound to plasma proteins in vivo (41), this is saturatable within the therapeutic range, suggesting VPA concentrations that inhibit HDAC activity are achievable in vivo (ref. 51 and references therein). VPA also possesses favorable pharmacokinetic properties (37), has been in clinical use for over 35 years, and is generally well tolerated (41). VPA has been shown to induce differentiation of leukemic cells (37) and cell cycle arrest of glioma cells (39) and to inhibit angiogenesis (56). It will be interesting to determine whether VPA possesses cancer chemopreventive properties. Future studies can be done to investigate whether long-term use of VPA in the treatment of epilepsy correlates with reduced cancer incidence. Such a study may also clarify whether additional agents, such as retinoids, may be required to enhance the ability of VPA to promote cellular differentiation and reduce cancer incidence.
Retinoids have already been used in the treatment of acute promyelocytic leukemia (20, 21) and combination therapies, including retinoids, are being investigated for use in the treatment of breast cancers (57). Although the exact mechanisms by which retinoids inhibit cancer progression are not fully understood, they are believed to inhibit mitogen signaling (58) and to induce multiple downstream signaling pathways (59), which contribute to the apoptosis (60), growth arrest (22), and/or differentiation (20) of cancer cells. It is believed the proapoptotic actions of RA are modulated by RARβ2 in breast cancer cells (25) and there is evidence that RARβ2 influences cellular levels of important cell cycle regulators, including p21CIP1 (61) and p27KIP1 (62). Therefore, reduced expression of RARβ2 results in the loss of multiple downstream growth inhibitory pathways and resistance to RA-dependent growth inhibition (27).
This study reveals for the first time that VPA, in combination with RA and Aza-dC, relieves the epigenetic repression of the RARβ2 tumor suppressor gene in MCF-7 breast cancer cells. MCF-7 cells represent a model of comparatively well-differentiated mammary carcinoma in which to investigate the mechanism of action of VPA at a prototypical silenced tumor suppressor promoter (RARβ2-RARE). Our data indicate that VPA, in combination with RA and Aza-dC, can restore RARβ2 expression and histone H3 acetylation to the silenced RARβ2-RARE in MCF-7 cells. However, VPA, in the absence of Aza-dC, was unable to induce robust transcriptional activation at the RARβ2-RARE (Fig. 1). This is consistent with a previous study (6) and suggests that concomitant inhibition of HDACs and DNMTs is required to overcome the epigenetic barrier to RA-dependent transactivation at the RARβ2-RARE in MCF-7 breast cancer cells. As RARβ2 was the only RA receptor that was not constitutively expressed in the breast cancer cells included in this study (Fig. 2), we next examined the ability of VPA to restore and enhance expression of RARβ2 in distinct breast cancer cell types. In MCF-7, MDA-MB-453, and SK-BR3 cells, the combined use of VPA with RA and Aza-dC resulted in the greatest enhancement of RARβ2 expression (Fig. 3).
Consistent with the hypothesis that RARβ2 is an important mediator of the potent antiproliferative and cancer chemopreventive properties of RA (22, 24, 25, 31), the reactivation of RARβ2 in MCF-7 cells (Fig. 3A) did coincide with growth inhibition (Fig. 4C). However, significant growth inhibition was also observed in MCF-7 (Figs. 4A and B and 5B) and MDA-MB-231 (Fig. 4D and E) cells treated with drug combinations that failed to enhance RARβ2 expression when quantified following 96-hour drug treatment (Fig. 3A and B). We next examined whether drug combinations employing VPA concentrations (250-50 μmol/L) lower than those reported in epilepsy patients (51, 52) enhanced the restoration of expression of RARβ2 (Fig. 5A, C, E, and G) and inhibited proliferation of a range of breast cancer cells (Fig. 5B, D, F, and H). Expression of RARβ2 was restored in MDA-MB-231 (Fig. 5B), MDA-MB-453 (Fig. 5E), and SK-BR3 (Fig. 5G) cells treated with RA, Aza-dC, and VPA (250-0 μmol/L) and each drug combination resulted in similar growth inhibition (Fig. 5D, F, and H). Under the conditions employed, trace expression of RARβ2 was detected in MCF-7 cells treated with RA and Aza-dC (Figs. 1A and 5A). A high level of RARβ2 expression was detected in MCF-7 cells treated with RA, Aza-dC, and VPA (250-50 μmol/L; Fig. 5A). However, similar growth inhibition was observed for each drug combination examined (Fig. 5B). It is possible that early but transient restoration of RARβ2 expression accounts for this growth inhibition observed in the absence of strong RARβ2 expression at 96 hours. However, it is likely these epigenetic drug combinations promote many RARβ2-independent growth regulatory pathways (5, 6), which contribute to the growth inhibition observed in the absence of increased RARβ2 expression.
Conversely, MDA-MB-231 treated with RA and VPA express RARβ2 (Fig. 3B) but remain resistant to growth inhibition (Fig. 4D). This is analogous to the HS578T breast cancer cell line, which expresses RARβ2 constitutively (Fig. 2E; ref. 27) yet is resistant to the growth inhibitory actions of retinoids (27). Although several previous reports have implied a crucial role for RARβ2 in mediating the antiproliferative actions of RA (23, 24, 34, 63, 64), it is evident from this and other studies (62, 65, 66) that other genes are necessary to induce cell cycle arrest and/or sensitize cancer cells to the growth inhibitory pathways regulated by retinoids (59). However, restoration of retinoid signaling and RARβ2 expression, in particular, represents an important contribution toward inhibiting breast cancer proliferation (23, 34). Indeed, recent studies are beginning to delineate the specific functions of the diverse pathways that mediate the growth inhibitory effects induced by retinoids and chromatin-targeted drugs. It was shown recently that growth inhibitory pathways induced by retinoids, HDAC inhibitors, and IFN-γ converge via the tumor necrosis factor–related apoptosis-inducing ligand proapoptotic pathway (60). Therefore, combination therapies, which target the epigenetic status of multiple tumor suppressor and growth regulatory genes and their downstream pathways (5, 6, 9, 14, 60), may be most effective in sensitizing a range of breast and other solid tumors to the growth inhibitory actions of retinoids.
In conclusion, we have shown that VPA, with RA and Aza-dC, can contribute to the reactivation and enhancement of expression of a prototypical silenced tumor suppressor gene (RARβ2) in breast cancer cells. It is therefore possible that combination therapies employing VPA will be useful in the treatment or chemoprevention of breast cancers. Although VPA has already shown considerable promise in inhibiting endometrial cell growth in nude mice (38) and diminishing adenoma formation in APCmin mice (40), it will be necessary to confirm the efficacy of VPA, in combination with therapeutic concentrations of RA and DNMT inhibitors, in a breast cancer mouse xenograft study. Such experiments will be of increasing clinical relevance when done with new classes of DNMT inhibitors (16, 67, 68), which are currently undergoing clinical trials.
Grant support: NIH grants R01DE10389 and R01CA77509 (L.J. Gudas) and AACR-Cancer Research Foundation of America postdoctoral fellowship (N.P. Mongan).
Revision received December 22, 2004.
American Association for Cancer Research
Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF. Histone deacetylases: unique players in shaping the epigenetic histone code. Ann N Y Acad Sci 2003;983:84–100.
Robertson KD, Uzvolgyi E, Liang G, et al. The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 1999;27:2291–8.
Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415–28.
Kelly WK, O'Connor OA, Marks PA. Histone deactylase inhibitors: from target to clinical trials. Expert Opin Investig Drugs 2002;11:1695–713.
Kim MS, Kwon HJ, Lee YM, et al. Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med 2001;7:437–43.
Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999;21:103–7.
Mistsiades CS, Mitsiades NS, McMullan CJ, et al. Transcriptional signature of histone deactylase inhibition in multiple myeloma: biological and clinical implications. Proc Natl Acad Sci U S A 2004;101:540–5.
Tang R, Faussat AM, Majdak P, et al. Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1. Leukemia 2004;18:1246–51.
Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE. Synergistic activation of functional estrogen receptor (ER)-α by DNA methyltransferase and histone deacetylase inhibition in human ER-α negative breast cancer cells. Cancer Res 2001;61:7025–9.
Sirchia SM, Ferguson AT, Sironi E, et al. Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor β2 promoter in breast cancer cells. Oncogene 2000;19:1556–63.
Arapshian A, Kuppumbatti YS, Mira-y-Lopez R. Methylation of conserved CpG sites neighboring the β retinoic acid response element may mediate retinoic acid receptor β gene silencing in MCF-7 breast cancer cells. Oncogene 2000;19:4066–70.
Yang Q, Mori I, Shan L, et al. Biallelic inactivation of retinoic acid receptor β2 gene by epigenetic change in breast cancer. Am J Pathol 2001;158:299–303.
Kim TY, Jong HS, Song SH, et al. Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanisms in gastric cancer cells. Oncogene 2003;22:3943–51.
Farias EF, Arapshian A, Bleiweiss IJ, Waxman S, Zelent A, Mira-y-Lopez R. Retinoic acid receptor α2 is a growth suppressor epigenetically silenced in MCF-7 human breast cancer cells. Cell Growth Differ 2002;13:335–41.
Gilbert J, Gore SD, Herman JG, Carducci MA. The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res 2004;10:4589–96.
Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004;429:457–63.
Warrell RP Jr, He LZ, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst 1998;90:1621–5.
Munster PN, Troso-Sandoval T, Rosen N, Rifkind R, Marks PA, Richon VM. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells. Cancer Res 2001;61:8492–7.
Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J 1996;10:940–54.
Warrell R Jr, Frankel S, Miller W, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans retinoic acid). N Engl J Med 1991;324:1385–93.
Petti MC, Fazi F, Gentile M, et al. Complete remission through blast cell differentiation in PLZF-RARα-positive acute promyelocytic leukemia: in vitro and in vivo studies. Blood 2002;100:1065–7.
Seewaldt VL, Johnson BS, Parker MB, Collins SJ, Swisshelm K. Expression of retinoic acid receptor β mediates retinoic acid induced growth arrest and apoptosis in breast cancer cells. Cell Growth Differ 1995;6:1077–88.
Sirchia SM, Ren M, Pili R, et al. Endogenous reactivation of the RAR β2 tumor suppressor gene epigenetically silenced in breast cancer. Cancer Res 2002;62:2455–61.
Faria TN, Mendelsohn C, Chambon P, Gudas LJ. The targeted disruption of both alleles of RARβ2 in F9 cells results in the loss of retinoic acid-associated growth arrest. J Biol Chem 1999;274:26783–8.
Liu Y, Lee MO, Wang HG, et al. Retinoic acid receptor β mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells. Mol Cell Biol 1996;16:1138–49.
Chen CF, Goyette P, Lohnes D. RARγ acts as a tumor suppressor in mouse keratinocytes. Oncogene 2004;23:5350–9.
van der Burg B, van der Leede BM, Kwakkenbos-Isbrucker L, Salverda S, de Laat SW, van der Saag PT. Retinoic acid resistance of estradiol-independent breast cancer cells coincides with diminished retinoic acid receptor function. Mol Cell Endocrinol 1993;91:149–57.
Suh YA, Lee HY, Virmani A, et al. Loss of retinoic acid receptorβ gene expression is linked to aberrant histone H3 acetylation in lung cancer cell lines. Cancer Res 2002;62:3945–9.
Nakayama T, Watanabe M, Yamanaka M, et al. The role of epigenetic modifications in retinoic acid receptor β2 gene expression in human prostate cancers. Lab Invest 2001;81:1049–57.
Hayashi K, Yokozaki H, Goodison S, et al. Inactivation of retinoic acid receptor β by promoter CpG hypermethylation in gastric cancer. Differentiation 2001;68:13–21.
Xu XC, Sneige N, Liu X, et al. Progressive decrease in nuclear retinoic acid receptor β messenger RNA level during breast carcinogenesis. Cancer Res 1997;57:4992–6.
de The H, Vivanco-Ruiz MM, Tiollais P, Stunnenberg H, Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor β gene. Nature 1990;343:177–80.
Jeronimo C, Henrique R, Hoque MO, et al. Quantitative RARβ2 hypermethylation: a promising prostate cancer marker. Clin Cancer Res 2004;10:4010–4.
Lin B, Chen GQ, Xiao D, et al. Orphan receptor COUP-TF is required for induction of retinoic acid receptor β, growth inhibition and apoptosis by retinoic acid in cancer cells. Mol Cell Biol 2000;20:957–70.
Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 2001;276:36734–41.
Gurvich N, Tsygankova OM, Meinkoth JL, Klein PS. Histone deactylase is a target of valproic acid mediated cellular differentiation. Cancer Res 2004;64:1079–86.
Göttlicher M, Minucci S, Zhu P, et al. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J 2001;20:6969–78.
Takai N, Desmond JC, Kumagai T, et al. Histone deacetylase inhibitors have a profound antigrowth activity in endometrial cells. Clin Cancer Res 2004;10:1141–9.
Bacon CL, Gallagher HC, Haughey JC, Regan CM. Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase. J Neurochem 2002;83:12–9.
Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Göttlicher M. Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis. Cancer Cell 2004;5:455–63.
Perucca E. Pharmacological and therapeutic properties of valproate. CNS Drugs 2002;16:695–714.
Kündgen A, Strupp C, Aivado M, et al. Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-trans retinoic acid. Blood 2004;104:1266–9.
Di Croce L, Raker VA, Corsaro M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 2002;295:1079–82.
Virmani AK, Rathi A, Zochbauer-Muller S, et al. Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas. J Natl Cancer Inst 2000;92:1303–7.
Jing Y, Zhang J, Bleiweiss IJ, Waxman S, Zelent A, Mira YLR. Defective expression of cellular retinol binding protein type I and retinoic acid receptors α2, β2, and γ2 in human breast cancer cells. FASEB J 1996;10:1064–70.
Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 2000;103:843–52.
Andreola F, Giandomenico V, Spero R, De Luca LM. Expression of a smaller lecithin:retinol acyl transferase transcript and reduced retinol esterification in MCF-7 cells. Biochem Biophys Res Commun 2000;279:920–4.
Guo X, Ruiz A, Rando RR, Bok D, Gudas LJ. Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines. Carcinogenesis 2000;21:1925–33.
Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001;25:402–8.
Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 2000;285:194–204.
El Desoky ES, Fuseau E, El Din Amry S, Cosson V. Pharmacokinetic modelling of valproic acid from routine clinical data in Egyptian epileptic patients. Eur J Clin Pharmacol 2004;59:783–90.
Besag FM, Berry DJ, Vasey M. Methsuximide reduces valproic acid serum levels. Ther Drug Monit 2001;23:694–7.
Cinatl J Jr, Cinatl J, Driever PH, et al. Sodium valproate inhibits in vivo growth of human neuroblastoma cells. Anticancer Drugs 1997;8:958–63.
Glaser KB, Staver MJ, Waring JF, Stender J, Ulrich RG, Davidsen SK. Gene expression profiling of multiple histone deactylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2003;2:151–63.
Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002;1:287–99.
Michaelis M, Michaelis UR, Fleming I, et al. Valproic acid inhibits angiogenesis in vitro and in vivo. Mol Pharmacol 2004;65:520–7.
Toma S, Raffo P, Nicolo G, et al. Biological activity of all-trans-retinoic acid with and without tamoxifen and α-interferon 2a in breast cancer patients. Int J Oncol 2000;17:991–1000.
Tighe AP, Talmage DA. Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling. Exp Cell Res 2004;301:147–57.
Niles RM. Signaling pathways in retinoid chemoprevention and treatment of cancer. Mutat Res 2004;555:81–96.
Clarke N, Jimenez-Lara AM, Voltz E, Gronemeyer H. Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL. EMBO J 2004;23:3051–60.
Suzui M, Shimizu M, Masuda M, Lim JTE, Yoshimi N, Weinstein IB. Acyclic retinoid activates retinoic acid receptor β2 and induces transcriptional activation of p21CIP1 in HepG2 human hepatoma cells. Mol Cancer Ther 2004;3:309–16.
Li R, Faria TN, Boehm M, Nabel EG, Gudas LJ. Retinoic acid causes cell growth arrest and an increase in p27 in F9 wild type but not in F9 retinoic acid receptor β2 knockout cells. Exp Cell Res 2004;294:290–300.
Dietze EC, Caldwell LE, Marcom K, et al. Retinoids and retinoic acid receptors regulate growth arrest and apoptosis in human mammary epithelial cells and modulate expression of CBP/p300. Microsc Res Tech 2002;59:23–40.
Lin F, Xiao D, Kolluri SK, Zhang X. Unique anti-activator protein-1 activity of retinoic acid receptor β. Cancer Res 2000;60:3271–80.
Cho Y, Talmage DA. Protein kinase Cα expression confers retinoic acid sensitivity on MDA-MB-231 human breast cancer cells. Exp Cell Res 2001;269:97–108.
Cho Y, Tighe AP, Talmage DA. Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase C α expression and activity. J Cell Physiol 1997;172:306–13.
Goffin J, Eisenhauer E. DNA methyltransferase inhibitors—state of the art. Ann Oncol 2002;13:1699–716.
Stewart DJ, Donehower RC, Eisenhauer EA, et al. A phase I pharmacokinetic and pharmacodynamic study of the DNA methyltransferase 1 inhibitor MG98 administered twice weekly. Ann Oncol 2003;14:766–74.
Thank you for sharing this Molecular Cancer Therapeutics article.
You are going to email the following Valproic acid, in combination with all-trans retinoic acid and 5-aza-2′-deoxycytidine, restores expression of silenced RARβ2 in breast cancer cells
Message Subject (Your Name) has forwarded a page to you from Molecular Cancer Therapeutics
Message Body (Your Name) thought you would be interested in this article in Molecular Cancer Therapeutics.
Mol Cancer Ther March 1 2005 (4) (3) 477-486; DOI: 10.1158/1535-7163.MCT-04-0079
Prediction of individual response to platinum/paclitaxel combination using novel marker genes in ovarian cancers
Low doses of cisplatin or gemcitabine plus Photofrin/photodynamic therapy: Disjointed cell cycle phase-related activity accounts for synergistic outcome in metastatic non–small cell lung cancer cells (H1299)
Semisynthetic homoharringtonine induces apoptosis via inhibition of protein synthesis and triggers rapid myeloid cell leukemia-1 down-regulation in myeloid leukemia cells
Show more Article
About MCT
Copyright 2016 by the American Association for Cancer Research.

redpajama_set_name: RedPajamaCommonCrawl


Whether you're a small business looking to put word out about your products, or a multinational company that is looking to expand into new markets, the same basic, underlying internet marketing strategy applying either case- allowing people to reach you, and interact with you instantly and transparently.
At SocialSpace Global Media, we specialize in cutting-edge internet marketing services (social media marketing, email marketing, mobile marketing, SEO, online advertising, ebranding, etc) according to your needs and targets in order to achieve an enhancement of interaction between yourself and your customers in the most uninhibited way possible. We don't just set you up with a media campaign and leave you with no guidance afterwards, together we follow every step of the way, ensuring that word gets out and your products or services are among the best in town! To add with we've got special prizes available for every company's budget! So stop worrying about the cost of internet marketing services, and start imagining the enormous benefits that you stand to reap from making people capable of connecting with you instantly, no matter what the time or place.


text:
Q: Is there a way to list all python virtual environments created using the venv module? Conda allows me to list all virtual environments as shown here. The commands are:
conda info --envs OR conda env list

I want to do that using pip. Does pip have any option to list all virtual environments created by me ? I have created a virtual environment on my desktop but I cannot figure out a way to list it along with the base environment.

A: No, not in an easy way.
Python virtual environments created with venv, virtualenv and most other python-only virtual environments can be stored anywhere on the disk.  And AFAIK they are not indexed, they are truly isolated (after all you can just remove venv directory and be done with it, you don't need to do anything special). They are also unmanaged by an environment manager.  So that would require entire disk scan. Which potentially can be done (you can search for all Python executables for example) but is rather painful.
It works with miniconda because miniconda manages other packages and files that it installs, so it places venvs in concrete path, e.g. /home/username/miniconda/envs/.

A: One indirect way is to run below command and see the directories where the python executable resides. Some of the paths will be python virtual environments and you can verify it by listing the files in those paths.
$ whereis python

A: You can use conda to create and manage venv and virtualenv environments and other packages installed using pip.
First create a conda environment with CONDA AND PIP installed into it, e.g.,
conda create --name core --channel conda-forge python=3.9 conda pip

Here I created the conda environment named "core" and installed Python 3.9, conda, and pip into it.  So now the 'core' conda environment functions like an administrative environment shell.  By installing conda into the conda environment, conda will track packages installed by pip into that environment.  You must use "pip install" INSIDE this new conda environment, so conda will index and track those pip package installations.  However, conda will still not index and centrally manage the venv environments, like it does for its own conda environments.
Here is a very good detailed guide that explains how and why to use conda and pip for virtual environments.  It covers the important aspect of using conda and pip together.
Why You Need Python Environments and How to Manage Them with Conda
Create virtual environments for python with conda


text:
Incentives to share: why copyright continues to play a role online
posted October 28, 2013 by Terry Hart.
Fred von Lohmann, former staff attorney of the EFF and currently Legal Director of Copyright at Google, has been presenting a talk recently on "Revising the Copyright Act for the 21st Century." While the views Lohmann expresses in the talk may not necessarily reflect the views of Google, it is reasonable to assume that there is close alignment.
The talk is motivated by review efforts that have begun in earnest by various government entities in recent months: the House Judiciary IP Subcommittee, the Copyright Office, and the Internet Policy Task Force in the Department of Commerce. In it, Lohmann makes a range of points about the current state of copyright and its future; while I haven't been able to find a video or transcript of the talk, the Yale Information Society Project (among others) livetweeted an October 15th presentation, which seems to be fairly comprehensive.
Lohmann calls for an aggressive reshaping of copyright law, not only domestically but internationally as well. Many of the fundamental changes would clearly benefit large online service providers like Lohmann's employer; very little is said about ensuring that individual creators have meaningful rights that enable them to build sustainable creative careers.
One of Lohmann's major points is that "Copyright is badly 'fit for purpose.'" The law "assumes that full rights apply to all works, even given the sheer quantity of creativity enabled by digital tools." According to Lohmann, "Ninety-nine percent of copyrighted works today are not incentivized by copyright." Lohmann suggests that YouTube and Instagram users, for example, aren't incentivized by the law's specific features, such as its duration or reversion rights. He asks, "Is [the] classic copyright incentive story necessary or appropriate for all these categories of works?"
Lohmann's question is premised on the flawed claim that the sole reason for securing copyright is to incentivize creation. That is only part of the reason for copyright. Just as important – if not more so – is the role copyright plays in providing incentives to publish and disseminate the works that authors create.
The Supreme Court made this crystal clear less than two years ago in Golan v. Holder. "Nothing in the text of the Copyright Clause confines the 'Progress of Science' exclusively to 'incentives for creation.' Evidence from the founding, moreover, suggests that inducing dissemination—as opposed to creation—was viewed as an appropriate means to promote science." The Southern District of New York echoed this idea last summer. "There is a strong public interest in the copyright system's function of motivating individuals to make available their creative works and increase the store of public knowledge." 1ABC v Aereo, No. 12-Civ-1540(AJN), order denying preliminary injunction (SDNY, July 11, 2012).
It may seem odd to suggest to anyone who has spent more than five minutes on social networking sites to suggest that incentives are needed for users to share things publicly. But much more is needed beyond the design of a particular social networking service to make it widely used or widely ignored – and some services, such as Myspace and Digg, have seen just how quickly a service can go from the former to the latter.
There are many factors that individuals and internet uses consider when making the choice of what and when to share something online. Most often, these are noneconomic considerations.
Barely a week goes by without a new privacy or term change that draws outrage. Earlier this month, Google announced that it would be "placing the photos, profiles and comments of some users in advertising endorsements across the Web." In September, Facebook released a new "Statement of Rights and Responsibilities" policy that caused the FTC to begin an inquiry (Facebook is governed by a 2011 agreement with the FTC that requires it to get explicit consent of its users before making material changes to its privacy policy). As Mashable reported, "Most alarming to users was the understanding that their profile data, including their profile picture, name, and personal information, could show up as part of a Facebook ad their friends may see on the site." And nearly a year ago, Instagram faced consumer ire over terms that would let it commercialize photos uploaded by users.
Sometimes it's not even an actual change but the perception of change that will cause users to think twice about disseminating their creative work. This past spring, there were reports that users were fleeing Tumblr once it was announced that the service would be purchased by Yahoo. Some have suggested that the motivation over leaving (or threatening to leave) the free service were concerns that Yahoo would change the characteristic of the community, tighten content standards, or overrun the site with advertisting.
The outcry that accompanies such announcements demonstrates quite clearly that many users factor into their decision to share photos, comments, and other content what will happen once its uploaded. Maintaining some control serves as an incentive to sharing in the first place. And if we want to encourage such dissemination, we need to continue to recognize that individuals have rights to their expression.
A large amount of those who share personal photos, writing, thoughts and other creative expression on social networking sites don't typically look to commercialize such expression. Likewise, they rarely need incentives to create such expression. But they do have some expectation when disseminating their expression that their work won't appear in advertising, as stock imagery for political causes they might not agree with, as endorsing some brand or cause. In other words, maintaining some level of choice and control over our stuff gives us an incentive to share our stuff.
Of course, much of these issues are governed by a particular site's terms of service or privacy policy. But these agreements are not enough. They can mediate the relationship between user and service, and users are able to make their own decisions about where to upload based on what terms are acceptable – though much more could be done to make such terms more transparent and understandable as well as address the issue of how easily services can change terms subsequently.
But contracts only go so far – terms of service and contracts don't extend to non-contracting third parties. Copyright serves as a backstop to what non-contracting parties can do. It's not the only mechanism, of course; various torts and statutes may govern what third parties can do with users' expression. But it certainly plays an important role. Again, copyright doesn't force anyone into sharing or not sharing their expression. It just prevents corporations and other parties from making that choice for them.
What's more, copyright reinforces First Amendment values that may come into play. Not only does it "motivat[e] individuals to make available their creative works and increase the store of public knowledge," but it also protects the freedom not to speak—a necessary complement to the freedom to speak. 2Harper & Row, Publishers, Inc. v. Nation Enterprises, 471 US 539, 560 (1985).
Copyright aligns with internet users' free speech rights and their expectations about what should happen with their photos, writing, and other expression once it is made available to the public online. We shouldn't be in such a rush to declare it a "bad fit" and start thinking of ways to toss it aside or turn it into something that only benefits large internet companies at the expense of the public. That's not to say that there are specific areas of the law that could be made better, but the basic principles of copyright law are just as applicable and vital today to the 99% of creators today.
1. ↑ ABC v Aereo, No. 12-Civ-1540(AJN), order denying preliminary injunction (SDNY, July 11, 2012).
2. ↑ Harper & Row, Publishers, Inc. v. Nation Enterprises, 471 US 539, 560 (1985).
John Warr
Google and big tech companies don't give a damn about "meaningful rights for creators" that allow them "to build sustainable creative careers." There is more recorded music already out there to satisfy any ones needs, if no more was created it wouldn't affect big tech in the slightest, as people could spend a life time listening to 'new' old stuff without getting bored. The same is true of films and books. All of that stuff can be monetarised by big tech, whether any more ever gets created is neither here nor there.
Good point. The idea that there is already too much stuff being created is rarely raised. There is too much content available and it is way beyond the capabilities of any human to consume even a tiny fraction of. We are at the point of extreme "information overload" as a society.
James_J
True, but there IS a large difference between the vast majority of crap out there and [quality] stuff that you'd actually like to see/hear. I think it's not that there's 'overload', it is just that the signal to noise ratio is getting worse…
Brett Glass
EFF is a lobbying shop for Google, so when von Lohmann left for his employer's employer he really did not change jobs. His goal is the same: to deprecate copyright so that Google can make even more money than it does already by violating authors' rights. (YouTube is the "go-to" site for piracy of music and video.)
ED STRAKER
Is Fred von Lohmann, Legal Director of Copyright at Google, ignoring the book "HR-2281: And Then the DMCA Didn't Apply on the Earth (Viacom vs. Google)", or even better, [he] never even heard about it. Oh, he knows. Button pushing power using digital files to destroy material objects instantly with no one knowing gives the man some power to spew to manipulate thought without an opposing view based on the computer science. For example:
As for copyright, there's no escape from Google/YouTube. First, they commit "willful blindness" and are subdirectory clones to the same URL. There is no DMCA Protection whether as a "service provider" (17 USC § 512(k)(1)(B), or not. If their subdirectories are specific URLs, then 80% are liable under 15 USC § 1125(d) (i.e.: the use of someone else's name without permission on a domain). Anonymous users? Then the signing of the Terms of Service is invalid (i.e.: an anonymous user does not have a signature). Repeat infringer policy (17 USC § 512(i)(1)(A)? An exclusive right is to authorize the reproduction (see 17 USC § 106), not a three strike rule with appended ads to reapply anonymously all over again.
Indeed, wouldn't 17 USC § 512(i)(1)(A) and an onus on copyright owners to find their works when infringed and depleted in seconds unconstitutional? Naturally Fred von Lohmann wants the Vampire Hunter version of copyright law. If it is not changed and catches up to Google (which it has now), Google goes down and has to admit lying, theft, and the imposed destruction of the US economy (and global economies) to destroy the right to sell physical objects.
Reality is sweet. Google knows the computer science while plaintiffs and [most] copyright owners do not. But now with Capital Records v. ReDigi (SDNY 2013), anything uploaded to YouTube is a copy, anything downloaded is a copy of that copy (see Page 5 of that ruling). And YouTube is on the record in their 3/11/2010 Memorandum of LAW in the Viacom case that no downloads occur (see Page 93, Line 9-12 signed by Andrew Shapiro). But downloads occur for all files + their TOS affirms the grant.
Isn't that an 18 USC § 1621 violation? To lie to a US Federal judge (and more judges)? Ask this, what keeps the United States lovely media to not state everything written above on-air? NSA monitoring? Sumner Redstone used the phrase "why should a farmer buy and raise cows when all the milk is free". I can go one step further. Why wholesale and retail Coca-Cola when the secret formula has been uploaded to YouTube as a copy, and downloaded as a copy of the copy, now all the anonymous users make coke in their kitchen and Coca-Cola (and the retailers) go out of business unaware the formula is downloaded 100,000,000 times in 2 weeks.
Paul Sweeting
Having read only the tweets you linked of Von Lohmann's speech I obviously can't say I have full knowledge of what he said (although I've known Fred professionally for years and have a pretty good sense of his views on the topic). But I don't think you are really making a counter-argument. I don't think Von Lohmann is arguing for the abolition of copyright, or that providing an incentive to create is its only aspect. He seems merely to be arguing that, insofar as providing an incentive to create is one part of the Copyright Act's design, it pretty self-evidently was not a major factor in motivating the creation of much of the content on social platforms today. Anyone who is at all familiar with the debates that have surrounded issues such as the length of the copyright term or reversion of rights, the two specific points from the speech you reference, knows that proponents of a longer term routinely assert it is necessary to provide an incentive to create. Von Lohmann is merely pointing to a body of evidence that suggests it isn't so.
Further, copyright is still very much in play when someone posts content to a social media platform. The terms of service of most such platforms, including Instagram, clearly stipulate that users retain ownership of the copyright in the content they post. As a condition of using the service they are required to sign over many of the rights that would normally obtain to copyrighted works, but that' no different in principle (although it may be in scope) to what record companies, publishers, syndicates and other copyright middlemen have been doing to creators since the dawn of copyright.
Totally agree. That's why I was sure this was intended as satire.
Miles Trenton
The idea that incentives are needed to publicly share is true, to a point. Everybody does something because they have a goal in mind – even if its just the positive feeling they get from supporting something they like. But some lower quality work needs more help to get shared online than others. Just the large number of companies listed at http://www.buyfacebooklikesreviews.com is proof of how often brands need to pay people to essentially share their creative work.
Everybody but the highest priced lawyers of big entertainment firms knows that copyright is beyond messed up, especially in an online age where remixes of great works only serve to promote the original work and benefits culture and everybody.


Norton antivirus program by Symantec is an excellent tool to detect and delete viruses from your computer by running a computer scan. To see if the computer is infected with the malicious programs it runs a computer scan. If any suspicious file is the detected during the scan, it deletes the file. Another good thing about Norton antivirus program is that every year its updated version is released. However, nothing is hundred percent fool-proofs in this world and Norton antivirus program is no exception. Sometimes Norton antivirus program may itself get infected with virus which has specifically been created to destroy the Norton antivirus program. If ever Norton antivirus program get attacked by a virus, it will be disabled and quick measures will have to be taken in order to restore Norton antivirus program on your computer. Do not worry! It is generally a simple process which demands not much effort on your behalf.
Simply follow the instructions given below to restore Norton antivirus program on your computer.
Step1: Go to the Norton website to run an online virus scan which will delete the virus affecting the Norton antivirus program from your computer.
Step 2: Uninstall Norton antivirus program from your computer. To do this, you need to go to the Start button present on the taskbar. A menu with different options will open. From this menu, choose Control Panel. Clicking on Control Panel will open the list of programs installed on your computer. Search for the Norton antivirus program in this list. Highlight it by clicking on Norton antivirus program. A Remove button will be seen. Start uninstalling the Norton antivirus program by clicking on this Remove button.
Step 3: After the Norton antivirus program is removed from your computer, restart the computer so that the changes made to the computer during the process may take effect.
Step 4: Now you need to reinstall the Norton antivirus program on your computer in order to keep protecting your computer from harmful programs. To reinstall the Norton antivirus program on your computer you will need its installation files. You will find this file either on a CD if you have purchased one or you will find the Norton antivirus installation files on the hard drive of your computer if you have downloaded the program from the Internet. Click on the installation file to open it.
Follow the instructions which comes on the screen for a smooth installation while installing the Norton antivirus program on your computer. If you encounter any trouble during the installation process, go for the support on the Symantec website. You can go for telephonic support, e-mail based support or chat based support provided by Symantec and ask for the instructions needed for the smooth installation of Norton antivirus program on your computer.
After you have installed the Norton antivirus program again on your computer, run a virus scan to be sure that your Norton antivirus program is working properly on your computer. Now you can keep using it without any hassle to protect your computer from viruses.


Welcome to the UHS adult travel program!
This travel program is open to: UHS faculty and former faculty; UHS alumni; UHS parents and former parents; and any related friends or family members of those with UHS connections.
Participants must be at least 21 years old at time of travel to join a tour.
For the 80 Day Club's inaugural trip we have decided to bring our friends, family, and members of the UHS community to one of our favorite cities - Rome, Italy. Rome is a beautiful and fascinating city with layers and layers of history, world class art, and awe-inspiring churches and architecture. We have chosen to spend a week in this magnificent city to allow for several exciting activities including visits to Vatican City, the Roman Colosseum and Forum, the Borghese Gallery, a wine-tasting in the Italian countryside, and a cooking class. There will also be considerable free time for participants to explore the various churches, museums, ruins, shops, and restaurants that Rome has to offer.
For more information, including specifics on pricing, itinerary, and other logistics, please visit our group tour site at www.grouptoursite.com/megtully.


A group of college kids travel to a small, mountain town called Madison County to interview the author of a tell-all book on the accounts of several grisly murders that happened there. But when the kids get to Madison County, the author is no where to be found and the towns people act like they haven't seen him in years. They also say that the killer never existed and the murders never happened. However, when the kids start digging around to get their own answers, they find out that the stories may be more real than the townspeople are letting on!
Regions are unavailable for this movie. Click the "Watch on Netflix" button to find out if Madison County is playing in your country.


text:
CRITIQUING THE CRITICS: CRITICS' CHOICE NOMINATIONS
The nominees for the 18th Annual Critics' Choice Movie Awards were announced. While many of their choices weren't surprising, it remains a mixed bag this year. Leading the pack was Spielberg's historical opus Lincoln with a record-setting 13 nominations.
Despite being a fantastic and ambitious film that includes Daniel Day Lewis in one of his best roles to date, one has to wonder whether or not it truly deserves to win best picture. At its core it remains a preachy, often dry film that does not speak to the masses. While its message of equality and peace is dead on, there are many that might find it less than entertaining. Still, it should be commended that such an intellectual film has received so much attention. While also of the same ilk, the upcoming Les Misérables followed close behind with 11 nominations. This one deserves a little more consideration. While it may be a story that has been remade dozens of times, the choice of using live music during the production means that the film audiences are watching is truly unlike any released before. It also seems to be taking a much grittier, more realistic approach than previous adaptations and should be commended on its ambition. Whether it is a good movie remains to be seen since it has not be released yet but this is defiantly one movie that deserves notice.
Other nominations included Life of Pi, Argo, Moonrise Kingdom and Zero Dark Thirty. "This has been a truly spectacular year in filmmaking and our voters had an embarrassment of riches to choose from," says BFCA President Joey Berlin. "To recognize the remarkable achievements across every genre of filmmaking we have added several new categories this year, including often overlooked performances in comedy and action. And to add even more fun, we're letting fans in on the voting in one special category."
The most confusing category seems to be for best animated feature. With nominees like Brave, Madagascar 3, Rise of the Guardians and Wreck-it-Ralph it seems like the judges just piled in every animated movie released this year without noticing if they had any real quality to them. Granted, movies like Brave and ParaNorman could both be considered groundbreaking and well deserving of honors. But Rise of the Guardians and Madagascar 3 seem to be thrown in there to appease the studios that produced them.
This year has shown us both the best and worst in cinema. One has to wonder what they were thinking when they nominated Ted as one of the best comedies of the year. At least gems like Looper, Cabin in the Woods and Prometheuswill finally get their due. A win for any of those films is a testament to good science fiction filmmaking.
The role of the critic is of course to find the best of the best. While the board that chose the nominees for this year's awards do not speak for all of us, their choices seem to be hitting the mark a good portion of the time. May the best film win!


text:
Holotrichia bakeri är en skalbaggsart som beskrevs av Moser 1924. Holotrichia bakeri ingår i släktet Holotrichia och familjen Melolonthidae. Inga underarter finns listade i Catalogue of Life.

Källor

Skalbaggar
bakeri

redpajama_set_name: RedPajamaWikipedia


text:
using System;
using System.Linq;
using System.Threading;
using System.Threading.Tasks;
using Microsoft.Azure;
using Microsoft.Azure.Management.Network.Models;

namespace Microsoft.Azure.Management.Network
{
    /// <summary>
    /// The Network Resource Provider API includes operations for managing the
    /// Virtual Networks for your subscription.
    /// </summary>
    public partial interface IVirtualNetworkOperations
    {
        /// <summary>
        /// The Put VirtualNetwork operation creates/updates a virtual network
        /// in the specified resource group.
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='virtualNetworkName'>
        /// The name of the virtual network.
        /// </param>
        /// <param name='parameters'>
        /// Parameters supplied to the create/update Virtual Network operation
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// Response for PutVirtualNetworks API service calls.
        /// </returns>
        Task<VirtualNetworkPutResponse> BeginCreateOrUpdatingAsync(string resourceGroupName, string virtualNetworkName, VirtualNetwork parameters, CancellationToken cancellationToken);

        /// <summary>
        /// The Delete VirtualNetwork operation deletes the specifed virtual
        /// network
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='virtualNetworkName'>
        /// The name of the virtual network.
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// If the resource provide needs to return an error to any operation,
        /// it should return the appropriate HTTP error code and a message
        /// body as can be seen below.The message should be localized per the
        /// Accept-Language header specified in the original request such
        /// thatit could be directly be exposed to users
        /// </returns>
        Task<UpdateOperationResponse> BeginDeletingAsync(string resourceGroupName, string virtualNetworkName, CancellationToken cancellationToken);

        /// <summary>
        /// The Put VirtualNetwork operation creates/updates a virtual
        /// networkin the specified resource group.
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='virtualNetworkName'>
        /// The name of the virtual network.
        /// </param>
        /// <param name='parameters'>
        /// Parameters supplied to the create/update Virtual Network operation
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// The response body contains the status of the specified asynchronous
        /// operation, indicating whether it has succeeded, is inprogress, or
        /// has failed. Note that this status is distinct from the HTTP status
        /// code returned for the Get Operation Status operation itself. If
        /// the asynchronous operation succeeded, the response body includes
        /// the HTTP status code for the successful request. If the
        /// asynchronous operation failed, the response body includes the HTTP
        /// status code for the failed request and error information regarding
        /// the failure.
        /// </returns>
        Task<AzureAsyncOperationResponse> CreateOrUpdateAsync(string resourceGroupName, string virtualNetworkName, VirtualNetwork parameters, CancellationToken cancellationToken);

        /// <summary>
        /// The Delete VirtualNetwork operation deletes the specifed virtual
        /// network
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='virtualNetworkName'>
        /// The name of the virtual network.
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// A standard service response including an HTTP status code and
        /// request ID.
        /// </returns>
        Task<AzureOperationResponse> DeleteAsync(string resourceGroupName, string virtualNetworkName, CancellationToken cancellationToken);

        /// <summary>
        /// The Get VirtualNetwork operation retrieves information about the
        /// specified virtual network.
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='virtualNetworkName'>
        /// The name of the virtual network.
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// Response for GetVirtualNetworks API service calls.
        /// </returns>
        Task<VirtualNetworkGetResponse> GetAsync(string resourceGroupName, string virtualNetworkName, CancellationToken cancellationToken);

        /// <summary>
        /// The list VirtualNetwork returns all Virtual Networks in a resource
        /// group
        /// </summary>
        /// <param name='resourceGroupName'>
        /// The name of the resource group.
        /// </param>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// Response for ListVirtualNetworks Api servive call
        /// </returns>
        Task<VirtualNetworkListResponse> ListAsync(string resourceGroupName, CancellationToken cancellationToken);

        /// <summary>
        /// The list VirtualNetwork returns all Virtual Networks in a
        /// subscription
        /// </summary>
        /// <param name='cancellationToken'>
        /// Cancellation token.
        /// </param>
        /// <returns>
        /// Response for ListVirtualNetworks Api servive call
        /// </returns>
        Task<VirtualNetworkListResponse> ListAllAsync(CancellationToken cancellationToken);
    }
}

redpajama_set_name: RedPajamaGithub


A breath of simplicity begets this natural look with the simple and modest design of the BARROW bottle. Purity of form and power of function blend together to create this timeless carafe. Perfect proportions ensure endless success.


text:
Giovanni Manzolini, né en 1700 à Bologne et mort le  dans la même ville, est un peintre et sculpteur néoclassique italien du  et anatomiste spécialisé dans les modèles céroplastiques anatomiques à l'Université de Bologne. Son épouse Anna Morandi est aussi une artiste céroplasticienne anatomique renommée.

Vie privée
Né en 1700, Giovanni Manzolini était fils de cordonnier. Il a aidé son père alors qu'il était enfant dans son métier. Il a marié en 1736 Anna Morandi, qu'il a connue de son enfance, qui avait aussi eu la formation d'artiste. Le couple a eu six enfants avant 1755, mais quatre d'entre eux sont morts en bas âge. Il est mort des complications de la tuberculose en 1755.

Carrière
Voulant devenir un peintre figuratif, Manzolini s'inscrit à l'âge de 40 ans au studio d'Ercole Lelli pour apprendre l'art de la sculpture anatomique. En 1743, il devient l'assistant de Lelli et sous ce dernier, il devient chef assistant par la commission pontificale pour la création de modèles en cire pour le musée de cire de l'Académie des sciences de l'institut de Bologne, position qu'il occuper pour trois années avant de démissionner vers la fin de l'année 1746 puisqu'il se sentait amer que Lelli l'avait privé de sa reconnaissance artistique pour sa grande connaissance de l'anatomie et de la sculpture anatomique.

Après s'être séparé de Lelli, il a ouvert son propre studio de modelage de cire et sa propre école d'anatomie. Sa femme a plus tard appris la dissection de cadavres, l'anatomie et l'art du modelage de cire pour pouvoire assister son époux et elle était la personne qui effectuer les conférences et les démonstrations à l'école de Manzolini. Le couple est peu après devenu renommé à travers l'Europe par divers anatomistes et artistes. Manzolini est aussi devenu professeur d'anatomie à l'Université de Bologne. Après la mort de son mari, Morandi est devenue conférencière à l'Université et est promue en tant que professeure en 1756.

Travaux
Manzolini a réalisé plusieurs modèles dont une collection d'organes sensoriels et vocaux destinés à la Royal Society de Londres, neuf modèles atomiques au Roi de Sardaigne et un recueil anatomique de 46 pièces de cire et de 125 pièces colorées en argile. Beaucoup d'entre eux ont été rachetés par Giovanni Antonio Galli pour servir de modèles pour son école de sages-femmes, puis rachetés par le Pape Benoît XIV et donnés à l'Académie des sciences de l'institut de Bologne pour être exposés au Palazzo Poggi, où ils sont encore à ce jour.

Article connexe
 Liste de peintres italiens
 Liste de sculpteurs

Notes et références

Liens externes

Peintre italien du XIXe siècle
Peintre néo-classique italien
Naissance en 1700
Naissance à Bologne
Décès en avril 1755
Décès à Bologne
Céroplasticien