Biomedical subjects

Peter B Dallas

Publications and source records attributed to Peter B Dallas.

8 recordsLinked to original sources

PubMed2005

The p270 (ARID1A/SMARCF1) subunit of mammalian SWI/SNF-related complexes is essential for normal cell cycle arrest.

Mammalian SWI/SNF-related complexes are ATPase-powered nucleosome remodeling assemblies crucial for proper development and tissue-specific gene expression. The ATPase activity of the complexes is also critical for tumor suppression. The complexes contain seven or more noncatalytic subunits; only one of which, hSNF5/Ini1/BAF47, has been individually identified as a tumor suppressor thus far. The noncatalytic subunits include p270/ARID1A, which is of particular interest because tissue array analysis corroborated by screening of tumor cell lines indicates that p270 may be deficient in as many as 30% of renal carcinomas and 10% of breast carcinomas. The complexes can also include an alternative ARID1B subunit, which is closely related to p270, but the product of an independent gene. The respective importance of p270 and ARID1B in the control of cell proliferation was explored here using a short interfering RNA approach and a cell system that permits analysis of differentiation-associated cell cycle arrest. The p270-depleted cells fail to undergo normal cell cycle arrest on induction, as evidenced by continued synthesis of DNA. These lines fail to show other characteristics typical of arrested cells, including up-regulation of p21 and down-regulation of cyclins. The requirement for p270 is evident separately in both the up-regulation of p21 and the down-regulation of E2F-responsive products. In contrast, the ARID1B-depleted lines behaved like the parental cells in these assays. Thus, p270-containing complexes are functionally distinct from ARID1B-containing complexes. These results provide a direct biological basis to support the implication from tumor tissue screens that deficiency of p270 plays a causative role in carcinogenesis.

3T3 Cells↗

PubMed2005

Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate?

BACKGROUND: The use of microarray technology to assess gene expression levels is now widespread in biology. The validation of microarray results using independent mRNA quantitation techniques remains a desirable element of any microarray experiment. To facilitate the comparison of microarray expression data between laboratories it is essential that validation methodologies be critically examined. We have assessed the correlation between expression scores obtained for 48 human genes using oligonucleotide microarrays and the expression levels for the same genes measured by quantitative real-time RT-PCR (qRT-PCR). RESULTS: Correlations with qRT-PCR data were obtained using microarray data that were processed using robust multi-array analysis (RMA) and the MAS 5.0 algorithm. Our results indicate that when identical transcripts are targeted by the two methods, correlations between qRT-PCR and microarray data are generally strong (r = 0.89). However, we observed poor correlations between qRT-PCR and RMA or MAS 5.0 normalized microarray data for 13% or 16% of genes, respectively. CONCLUSION: These results highlight the complementarity of oligonucleotide microarray and qRT-PCR technologies for validation of gene expression measurements, while emphasizing the continuing requirement for caution in interpreting gene expression data.

Algorithms↗

PubMed2005

Genomic deletions in cell lines derived from primitive neuroectodermal tumors of the central nervous system.

Extensive genomic deletions affecting a variety of chromosomes are a common finding in primitive neuroectodermal tumors of the central nervous system (CNS-PNETs), implicating the loss of multiple tumor suppressor genes in the pathogenesis of these tumors. We have used representational difference analysis, microsatellite mapping, and quantitative polymerase chain reaction to identify and verify the presence of genomic deletions on a number of chromosomes in CNS-PNET cell lines. This systematic approach has confirmed the importance of deletions at 10q, 16q, and 17p in PNET pathology and has revealed other regions of deletion not commonly described (e.g., Xq, 1p, 7p, and 13q). These data highlight the prevalence of hemizygous loss in CNS-PNET cells, suggesting that haploinsufficiency affecting multiple tumor suppressor genes may play a fundamental role in CNS-PNET pathogenesis. The identification of specific genes and signaling pathways that are compromised in CNS-PNET cells is crucial for development of more efficacious and less invasive treatments, as are urgently needed.

Cell Line, Tumor↗

PubMed2004

The murine cytomegalovirus M73.5 gene, a member of a 3' co-terminal alternatively spliced gene family, encodes the gp24 virion glycoprotein.

We have identified a novel family of five 3' co-terminal transcripts in murine cytomegalovirus (MCMV) arranged in a tail-to-tail orientation with respect to the MCMV glycoprotein H (gH) gene M75. They share the same exon 2 sequence but possess different exon 1 sequences. Two of these spliced transcripts (M73) encode the MCMV homolog of glycoprotein N (gN) entirely within exon 1. Two other transcripts designated M73.5 encode a previously described virion glycoprotein gp24 that shares its first 20 amino acids with gN, but which has another 64 amino acids encoded within exon 2. The fifth transcript, designated m60, has an 80-bp exon 1 near the MCMV oriLyt region 10.8 kb upstream of exon 2. Both MCMV M73.5 and m60 encode type II glycoproteins expressed at the cell surface. Their shared exon 2 coding sequences likely represent the highly conserved region of an as yet unidentified betaherpesvirus-specific glycoprotein species.

3T3 Cells↗

PubMed2004

Expression of p270 (ARID1A), a component of human SWI/SNF complexes, in human tumors.

Human SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromosomes and alter gene expression patterns. The activity of the complexes generally promotes tissue-specific gene expression and restricts cell proliferation. The ATPase that drives the complexes, BRG1, is essential for tumor suppression in mice and deficient in a variety of established human tumor cell lines. The complex contains at least 7 other core components, one of which is a large subunit designated p270. p270 RNA is expressed in all normal human tissues examined, but protein expression is severely reduced in at least 2 human tumor lines, C33A and T47D. We show here that loss of p270 in the C33A and T47D cell lines is evident at the RNA level as well as the protein level. The implication that p270 can be informatively screened at the RNA level made a high-efficiency cancer profiling array approach to screening human tumors feasible. Expression was screened in an array containing RNA-derived cDNA from 241 tumor and corresponding matched normal tissues from individual patients. p270 deficiency was observed at a higher overall frequency than BRG1 deficiency, but all tissues were not equally affected. Deficiency of p270 was observed most frequently in carcinomas of the breast and kidney. The results were most striking in kidney, where p270 expression was deficient in 30% of carcinoma samples screened. Screening of a panel of established human renal carcinoma-derived cell lines supports the frequency observed in the primary tumor tissue samples.

Breast Neoplasms↗

PubMed2004

Two related ARID family proteins are alternative subunits of human SWI/SNF complexes.

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.

Adenosine Triphosphatases↗

PubMed2004

The DNA-binding properties of the ARID-containing subunits of yeast and mammalian SWI/SNF complexes.

SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that are highly conserved from yeast to human. From yeast to human the complexes contain a subunit with an ARID (A-T-rich interaction domain) DNA-binding domain. In yeast this subunit is SWI1 and in human there are two closely related alternative subunits, p270 and ARID1B. We describe here a comparison of the DNA-binding properties of the yeast and human SWI/SNF ARID-containing subunits. We have determined that SWI1 is an unusual member of the ARID family in both its ARID sequence and in the fact that its DNA-binding affinity is weaker than that of other ARID family members, including its human counterparts, p270 and ARID1B. Sequence analysis and substitution mutagenesis reveals that the weak DNA-binding affinity of the SWI1 ARID is an intrinsic feature of its sequence, arising from specific variations in the major groove interaction site. In addition, this work confirms the finding that p270 binds DNA without regard to sequence specificity, excluding the possibility that the intrinsic role of the ARID is to recruit SWI/SNF complexes to specific promoter sequences. These results emphasize that care must be taken when comparing yeast and higher eukaryotic SWI/SNF complexes in terms of DNA-binding mechanisms.

Amino Acid Sequence↗

PubMed2002

ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development.

The ARID family of DNA binding proteins was first recognized approximately 5 years ago. The founding members, murine Bright and Drosophila dead ringer (Dri), were independently cloned on the basis of their ability to bind to AT-rich DNA sequences, although neither cDNA encoded a recognizable DNA binding domain. Mapping of the respective binding activities revealed a shared but previously unrecognized DNA binding domain, the consensus sequence of which extends across approximately 100 amino acids. This novel DNA binding domain was designated AT-rich interactive domain (ARID), based on the behavior of Bright and Dri. The consensus sequence occurs in 13 distinct human proteins and in proteins from all sequenced eukaryotic organisms. The majority of ARID-containing proteins were not cloned in the context of DNA binding activity, however, and their features as DNA binding proteins are only beginning to be investigated. The ARID region itself shows more diversity in structure and function than the highly conserved consensus sequence suggests. The basic structure appears to be a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of the basic six. It has also become apparent that the DNA binding activity of ARID-containing proteins is not necessarily sequence specific. What is consistent is the evidence that family members play vital roles in the regulation of development and/or tissue-specific gene expression. Inappropriate expression of ARID proteins is also increasingly implicated in human tumorigenesis. This review summarizes current knowledge about the structure and function of ARID family members, with a particular focus on the human proteins.

AT Rich Sequence↗