Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis.

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint is associated with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats, and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes, and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose, and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with P = e(-13) to e(-36) . Cell cycle expression networks show species, sex and tissue variability, and they are enriched in mRNA transcripts associated with mitosis, many of which are associated with chromosomal instability.

SAFB1 interacts with and suppresses the transcriptional activity of p53.

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.

Systems genetics analyses predict a transcription role for P2P-R: molecular confirmation that P2P-R is a transcriptional co-repressor.

BACKGROUND: The 250 kDa P2P-R protein (also known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations. RESULTS: GeneNetwork and associated gene ontology links were used to investigate the coexpression of P2P-R with distinct functional sets of genes in an adipocyte genetic reference panel of HXB/BXH recombinant strains of rats and an eye genetic reference panel of BXD recombinant inbred strains of mice. The results establish that biological networks of 75 and 135 transcription-associated gene products that include P2P-R are co-expressed in a genetically-defined manner in rat adipocytes and in the mouse eye, respectively. Of this large set of transcription-associated genes, >10% are associated with hormone-mediated transcription. Since it has been previously reported that P2P-R can bind the SRC-1 transcription co-regulatory factor (steroid receptor co-activator 1, [Ncoa1]), the possible effects of P2P-R on estrogen-induced transcription were evaluated. Estrogen-induced transcription was repressed 50-70% by the transient transfection of P2P-R plasmid constructs into four different cell types. In addition, knockdown of P2P-R expression using an antisense oligonucleotide increased estrogen-mediated transcription. Co-immunoprecipitation assays confirmed that P2P-R interacts with SRC-1 and also demonstrated that P2P-R interacts with estrogen receptor alpha. CONCLUSIONS: The findings presented in this study provide strong support for the value of systems genetics, especially GeneNetwork, in discovering new functions of genes that can be confirmed by molecular analysis. More specifically, these data provide evidence that the expression of P2P-R co-varies in a genetically-defined manner with large transcription networks and that P2P-R can function as a co-repressor of estrogen-dependent transcription.

De-differentiation-derived mesenchymal stem cells demonstrate selective repression in H19 bioregulatory RNA gene expression.

Cellular de-differentiation can induce anticancer activity that makes cells resistant to carcinogenesis, but the molecular mechanism of this phenomenon has not been defined. To determine whether stable molecular changes develop in association with the process of de-differentiation, DNA microarray analyses were performed. These analyses compared control undifferentiated cells with three carcinogenesis-resistant clones of de-differentiated cells that were derived from mature adipocytes. The results of analysis of 6,000 genes and 6,000 ESTs establish that relative to control cells, all three de-differentiation-derived cell clones demonstrate that only one gene shows a consistent difference in expression. The expression of the H19 bioregulatory RNA is repressed an average of >fourfold in all de-differentiated cell clones. Real-time PCR analyses confirm these findings. This suggests that decreased H19 expression may account, at least in part, for the anticancer activity observed in de-differentiated cell clones.

P2P-R expression is genetically coregulated with components of the translation machinery and with PUM2, a translational repressor that associates with the P2P-R mRNA.

P2P-R is a nuclear protein with potential functional roles in the control of gene expression and mitosis. The P2P-R protein also interacts with the p53 and Rb1 tumor suppressor proteins. To search for additional functional associations of P2P-R, we employed the WebQTL database that contains the results of cDNA microarray analysis on forebrain, cerebellum, and hematopoietic stem cell (HSC) specimens of multiple BXD recombinant inbred strains of mice. Using WebQTL, gene products were identified that show genetically based coexpression with P2P-R. Initial studies identified general groups of mRNAs that share common functional roles and high covariation in expression with P2P-R. These functional groups involved the regulation of transcription, nucleotide binding, translation control, and ion transport. The findings related to translational mechanisms were further evaluated. In HSCs, expression of P2P-R mRNA demonstrates an impressive expression correlation with a group of gene products associated with translation; high expression of P2P-R specifically was associated with decreased expression of 29 ribosomal protein mRNAs. In all three tissues that were screened using the WebQTL database, a strong positive expression covariance between P2P-R and the Pum2 gene product also was observed. PUM2 is a member of the highly conserved Puf family of RNA binding proteins that often function as gene-specific translation regulators. The ability of Puf proteins to repress translation is mediated by their binding to specific elements located in the 3' untranslated region (UTR) of their target mRNAs. To assess the functional significance of the strong genetic correlation in expression of P2P-R and PUM2, the 3' UTR of the P2P-R mRNA was analyzed and found to contain one perfect consensus and two near-perfect consensus PUM2 binding sequences. PUM2 pull-down methods combined with reverse transcription and RT-PCR confirmed that PUM2 does indeed bind P2P-R mRNA. These results suggest that P2P-R expression may be translationally regulated by PUM2 and that P2P-R may modulate translation by influencing ribosomal protein gene expression. This study represents the first description of a RNA target for mammalian Puf proteins and the first molecular confirmation of information obtained using the WebQTL database.

Functional potential of P2P-R: a role in the cell cycle and cell differentiation related to its interactions with proteins that bind to matrix associated regions of DNA?

P2P-R is the alternately spliced product of the P2P-R/PACT gene in that P2P-R lacks one exon encoding 34 amino acids. The 250 kDa P2P-R protein is the predominate product expressed in multiple murine cell lines. It is a highly basic protein that contains multiple domains including an N-terminal RING type zinc finger, a proline rich domain, an RS region, and a C-terminal lysine-rich domain. P2P-R binds the p53 and the Rb1 tumor suppressors and is phosphorylated by the cdc2 and SRPK1a protein kinases. P2P-R also interacts with scaffold attachment factor-B (SAF-B), a well characterized MARs (for matrix attachment regions) binding factor, and may interact with nucleolin, another MARs binding factor. In addition, P2P-R binds single strand DNA (ssDNA). The expression of P2P-R is regulated by differentiation and cell cycle events. P2P-R mRNA is markedly repressed during differentiation, whereas immunoreactive P2P-R protein levels are >10-fold higher in mitotic than in G(0) cells. The localization of P2P-R also is modulated during the cell cycle. During interphase, P2P-R is present primarily in nucleoli and nuclear speckles whereas during mitosis, P2P-R associates with the periphery of chromosomes. Overexpression of near full length P2P-R induces mitotic arrest in prometaphase and mitotic apoptosis, and overexpression of selected P2P-R segments also can promote apoptosis. This compendium of data supports the possibility that P2P-R may form complexes with the Rb1 and/or p53 tumor suppressors and MARs-related factors, in a cell cycle and cell differentiation-dependent manner, to influence gene transcription/expression and nuclear organization.

P2P-R protein overexpression restricts mitotic progression at prometaphase and promotes mitotic apoptosis.

Mitotic cells show a tenfold increase in immunoreactive P2P-R protein. During mitosis, the distribution of P2P-R protein also changes from a primary nucleolar localization in interphase cells to the periphery of chromosome in mitotic cells. These findings suggest that P2P-R might serve a functional role in mitosis. To test this possibility, human Saos2 cells were stably transfected with P2P-R DNA constructs and the biological effects of P2P-R overexpression were evaluated. Overexpression of near full-length P2P-R was found to have paradoxical effects on the relationship between proliferation and mitosis in the nine Saos2 cell clones that were studied. A significant repression in the population doubling rates was observed in all nine clones even though a significant increase in the frequency of easily detached cells with a mitotic morphology was apparent. Flow cytometric analysis confirmed that greater than two thirds of the cells with a mitotic morphology had a 4n DNA content. Confocal microscopy further established that 85% of the mitotic cell population had prometaphase characteristics suggesting that P2P-R overexpression restricts mitotic progression at prometaphase. Many cells with a mitotic morphology also showed signs of apoptosis with prominent cell surface blebs. Confocal microscopy confirmed that 25-40% of such mitotic cells were apoptotic with chromosomal abnormalities and cell surface blebbing. In association with mitotic apoptosis, P2P-R protein appears to dissociate from the periphery of chromosomes and localize in the cytoplasm and in cell surface blebs. The presence of P2P-R in cell surface blebs was confirmed by analysis of highly enriched populations of apoptotic cell surface blebs wherein Western blotting documented the presence of 250 kDa P2P-R. These results therefore suggest that P2P-R overexpression promotes both prometaphase arrest in mitosis and mitotic apoptosis.

P2P-R protein localizes to the nucleolus of interphase cells and the periphery of chromosomes in mitotic cells which show maximum P2P-R immunoreactivity.

P2P-R is a nuclear protein that can bind both p53 and Rb1. Its functions include roles in the control of RNA metabolism, apoptosis, and p53-dependent transcription. The expression of P2P-R also is repressed in G1 arrested terminally differentiated cells. The current studies therefore evaluated if P2P-R undergoes cell cycle-associated changes in its abundance and/or localization. Western blots show that relative to G0 quiescent cells, P2P-R protein levels are higher in populations of G2/M cells prepared by the physiological parasynchronization technique of serum deprivation followed by serum stimulation. More striking is the > 10-fold enrichment of P2P-R protein in specimens of highly purified mitotic cells prepared by the mitotic shake-select technique, or by synchrony with the mitotic spindle disruption agents nocodazole or vinblastine. These changes in P2P-R protein occur without a concomitant change in P2P-R mRNA expression suggesting that P2P-R immunoreactivity increases during mitosis. Confocal microscopy next established the localization of P2P-R to nucleoli in interphase cells and at the periphery of chromosomes in mitotic cells that lack nucleoli. The high levels of P2P-R localized to the periphery of chromosomes in mitotic cells suggest that P2P-R shares characteristics with other nucleolar proteins that associate with the periphery of chromosomes during mitosis. These include: nucleolin, B23, Ki67, and fibrillarin.

Traumatic hip dislocation in athletes.

Sports injuries are increasingly prevalent. Dislocations of the hip are rare; however, they are seen in high-velocity sports such as football and rugby. The injuries may be described as anterior or posterior, and classified according to severity of injury. It is critical to the sports medicine practitioner to promptly recognize and manage this complex injury. This article presents a functional management algorithm including reduction techniques for the simple hip dislocation. Early reduction minimizes complications in all studies. Immediate on-field reduction has been performed successfully and safely in a limited number of cases. An aggressive rehabilitation protocol is proposed to assist the athlete back to sporting activity as soon as possible. The potential benefits of early reduction limit the risk of avascular necrosis of the femoral head and sciatic neuropathy; therefore, further investigation into the standard use of this immediate reduction technique is warranted.