Retinoblastoma/p107/p130 pocket proteins: Protein dynamics and interactions with target gene promoters
ABSTRACT The retinoblastoma (RB) tumor suppressor and its family members, p107 and p130, function by repressing E2F transcription factor activity to limit the expression of genes required for cell cycle progression. Traditionally, it is thought that the RB family proteins repress E2F target gene expression through complexing with E2F at gene promoters. However, whereas chromatin immunoprecipitation experiments have demonstrated p107 and p130 at E2F-responsive promoters, RB chromatin association has not been reliably observed. Here we used green fluorescent protein-tagged proteins to rigorously explore the mechanism of RB-mediated transcriptional repression relative to its p107 and p130 family members. The use of live cell fluorescent imaging demonstrated that RB, p107, and p130 exhibit similar nuclear dynamics. Although these findings suggest a similar engagement with nuclear structures, chromatin immunoprecipitation approaches with multiple independent antibodies failed to detect the association of RB with target gene promoters. However, by employing antibodies directed against green fluorescent protein, we could utilize the same antibody to assess RB, p107, and p130 engagement. This approach demonstrated RB association with target gene promoters in a fashion analogous to p107 and p130. Extension of this technology demonstrated that direct RB phosphorylation disrupts promoter association to regulate transcription. Thus, RB is associated with promoters in a manner similar to p107/p130 and that association is modulated by phosphorylation during cell cycle progression.
- SourceAvailable from: Robert Clarke
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- "ChIP was carried out as previously described by Stengel et al. (2009). Briefly, cells were grown in the presence or absence of treatment, and cross-linked with formaldehyde . "
ABSTRACT: The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.Endocrine Related Cancer 03/2011; 18(3):333-45. DOI:10.1530/ERC-10-0262 · 4.91 Impact Factor
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- "Technical limitations have heretofore prevented systematic identification of pRb associated genes and proteins. While chromatin immunoprecipitation has been successfully used to identify pRb at predicted candidate genes, currently available antibodies have insufficient specificity for unbiased identification of pRb gene targets at the genome wide level(Stengel et al., 2009). Similarly, available pRb antibodies do not yield protein in sufficient yield or specificity for robust systematic identification of copurifying proteins. "
ABSTRACT: Loss of Rb1 tumor suppressor gene function is involved in the genesis of most human cancers. Novel therapies targeting Rb1 have been slow to develop because of our incomplete understanding of its molecular mechanisms of action. Rb1 protein (pRb) binds a host of cellular genes and proteins, and these molecular interactions mediate its various functions. Given the potential complexity of these molecular interactions and the lack of established methods for pRb purification, it has been difficult to systematically identify gene and protein interactions relevant to tumor suppression in different tissues in vivo. To address this limitation, we have generated a dual affinity tagged Rb1 allele in the mouse. The tagged allele functions as wild type and the encoded protein can be purified by tandem affinity chromatography. This allele will facilitate identification and characterization of native pRb molecular interactions in any tissue accessible in the mouse.genesis 01/2009; 48(2):121-6. DOI:10.1002/dvg.20589 · 2.04 Impact Factor
Conference Paper: Microwave radiation from a weakly non-Gaussian sea surface[Show abstract] [Hide abstract]
ABSTRACT: The small deviation from Gaussian statistics of the sea surface is introduced to describe the up and downwind difference in microwave brightness temperature. The effect of long wave asymmetry is estimated using small-slope expansion and model slope distribution. Numerical analysis shows that the long-wave asymmetry cannot explain the observed values of the first harmonic in azimuthal dependencies of microwave radiation. The distribution of ripples over the long waves is considered using small-slope approximation and hydrodynamic modulation transfer function. The comparison with experimental radiometric observations allows the authors to derive the imaginary part of the modulation transfer function, which is in reasonably good agreement with independent radar measurementsGeoscience and Remote Sensing Symposium Proceedings, 1998. IGARSS '98. 1998 IEEE International; 08/1998