Do breast cancer survivors increase their physical activity and enhance their health-related quality of life after attending community-based wellness workshops?

Many breast cancer survivors may be at increased risk for physical and psychological complications from cancer treatments. Research has shown that regular exercise can help ameliorate some of the lingering side effects of breast cancer treatments and improve health-related quality of life (HRQOL). Additionally, certain stress management techniques have helped increase HRQOL in breast cancer survivors. Few educational programs exist which address both the promotion of physical activity and use of mindfulness-based strategies to improve the health of breast cancer survivors. Community-based wellness workshops were designed to promote regular exercise and use of mindfulness-based techniques. There was an increase in physical activity and improvements on several HRQOL domains 1 month following the exercise workshops; although the results were not significant, they are encouraging.

Nuclear organization of pre-mRNA processing.

Recent studies have suggested that small nuclear ribonucleoprotein particles (snRNPs), non-snRNP splicing factors, and several heterogeneous nuclear RNP proteins change their organization within the cell in response to transcriptional activity. Several of the RNA substrates with which these factors interact have been shown to localize in tracks that are associated with regions in which splicing factors are concentrated (nuclear speckles). It is now thought that pre-mRNA splicing may occur within these tracks.

The cellular organization of gene expression.

Recent cell biological observations have provided new insights into how transcription, pre-mRNA splicing and 3' processing are organized and coordinated with each other in the mammalian cell nucleus. Morphological observations are supported by biochemical evidence that suggests physical interactions between components of the transcription and RNA processing machineries. A working model of the cellular organization of gene expression is now emerging.

Visualization of gene activity in living cells.

Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.

Neuro-modulation and bariatric surgery for type 2 diabetes mellitus.

Obesity has reached epidemic proportions and is continuing to grow into one of the leading healthcare issues worldwide. With this development, bariatric surgery has emerged as an acceptable treatment for morbid obesity, generally achieving meaningful and sustained weight loss. In a surprising turn of events, bariatric surgery was also found to be the most effective therapy for type 2 diabetes mellitus (T2DM). This observation has sparked a great deal of research that has improved our understanding of T2DM pathophysiology; it has facilitated the development of medical treatment and is expanding the indications for bariatric surgery. It was traditionally accepted that bariatric surgery causes weight loss by restriction of gastric volume, intestinal malabsorption, or a combination of the two. Laparoscopic adjustable gastric banding (LAGB) is considered a purely restrictive procedure that involves the placement of an adjustable band around the cardia of the stomach, creating a 15 ml pouch. Laparoscopic sleeve gastrectomy (LSG) is the resection of the fundus all along the greater curvature of the stomach. LSG was once considered a restrictive procedure, but this presumption has recently come under scrutiny. Bilio-pancreatic diversion (BPD) is an example of a procedure that was considered predominantly malabsorptive. In this operation, the ingested nutrients are diverted from the stomach to the ileum, bypassing a large segment of proximal bowel. Roux-en-Y gastric bypass (RYGB) traditionally combines both mechanisms, partitioning a small pouch from the proximal stomach and diverting the ingested nutrients to the jejunum with a roux-en-Y gastro-jejunostomy. However, recent investigation suggests additional mechanisms of action including hormonal. Today, RYGB is the procedure of choice for morbidly obese patients. The effect of bariatric surgery on T2DM was initially described in 1995 by Pories et al., who reported that there was an overall T2DM resolution after RYGB of 82.9% (1). A resolution rate of approximately 80% has been demonstrated repeatedly (2,3). The initial assumption was that the mechanism causing this effect was through weight loss. It is becoming evident that the anti-diabetic effect is not entirely weight loss as there is a consistent observation that the improvement of glucose and insulin levels occurs within days after RYGB, clearly too soon to be due to the weight loss (1,4). The ensuing body of literature has generated two leading theories attempting to explain this weight-independent anti-diabetic effect after RYGB. The 'hindgut' proposes that rapid delivery of partially digested nutrients to the distal bowel up-regulates the secretion of incretins such as glucagon-like peptide-1 (GLP-1). The result of the increased incretin secretion is an enhanced glucose-dependent insulin secretion, as well as a number of other changes causing improved glucose tolerance (4). In the second theory, 'the foregut hypothesis', the exclusion of the duodenum results in the inhibition of a 'putative' signal that is responsible for insulin resistance (IR) and/or abnormal glycaemic control. In a non-obese diabetic rat model, surgical diversion of the proximal bowel caused rapid improvement of diabetes without reduction of food intake or change in weight (5). Many aspects regarding surgical treatment of T2DM are still questionable and unexplained. Emerging data are starting to clarify the mechanisms participating in the anti-diabetic effect, and also challenging long-held theories.

Subversion of cell cycle regulatory pathways.

Human cytomegalovirus (HCMV) has evolved numerous strategies to commandeer the host cell for producing viral progeny. The virus manipulates host cell cycle pathways from the early stages of infection to stimulate viral DNA replication at the expense of cellular DNA synthesis. At the same time, cell cycle checkpoints are by-passed, preventing apoptosis and allowing sufficient time for the assembly of infectious virus.

Protein phosphorylation and the nuclear organization of pre-mRNA splicing.

Controlled execution of transcription and pre-mRNA splicing is crucial for proper gene expression. The organization of these essential events within the cell nucleus is only beginning to be understood. Here, we describe a model for the cellular arrangement of transcription and pre-mRNA splicing based on recent biochemical and morphological data: transcription and pre-mRNA splicing are spatially and temporally coordinated, and protein phosphorylation regulates both the activity and the subnuclear localization of pre-mRNA splicing factors in nuclear subcompartments.

Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription.

We have examined the nuclear localization of transiently and stably expressed nascent RNA transcripts containing or lacking introns in order to determine if the spatial association of RNA transcripts and pre-mRNA splicing factors in nuclei is random or functionally significant. Our findings show that the association between nascent RNA and splicing factors in the nucleus is intron-dependent when the RNAs are either transiently or stably expressed. Furthermore, our data indicate that splicing factors are recruited to the transcription sites. The presence of both pre-and mRNA at these locations suggest that pre-mRNA splicing occurs at these sites of transcription. In addition, electron microscopic examination of the highly active transcription sites has revealed a granular appearance which closely resembles, but is functionally different from, interchromatin granule clusters. Our findings demonstrate that the nucleus is highly organized and dynamic with regard to the functions of the transcription and pre-mRNA splicing.

Nonhistone protein BA is a glutathione S-transferase localized to interchromatinic regions of the cell nucleus.

A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.

Immunocytochemical localization of casein kinase II during interphase and mitosis.

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.

The dynamic organization of the perinucleolar compartment in the cell nucleus.

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181-1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471-11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring approximately 80-180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP-PTB fusion protein showed that at least three RRMs at either the COOH or NH2 terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.

Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity.

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.

In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

The perinucleolar compartment and transcription.

The perinucleolar compartment (PNC) is a unique nuclear structure localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III and two hnRNP proteins have been localized in the PNC (Ghetti, A., S. Piñol-Roma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181- 1193; Timchenko, L.T., J.W. Miller, N.A. Timchenko, D.R. DeVore, K.V. Datar, L. Lin, R. Roberts, C.T. Caskey, and M.S. Swanson. 1996. Nucleic Acids Res. 24: 4407-4414; Huang, S., T. Deerinck, M.H. Ellisman, and D.L. Spector. 1997. J. Cell Biol. 137:965-974). In this report, we show that the PNC incorporates Br-UTP and FITC-conjugated CTP within 5 min of pulse labeling. Selective inhibition of RNA polymerase I does not appreciably affect the nucleotide incorporation in the PNC. Inhibition of all RNA polymerases by actinomycin D blocks the incorporation completely, suggesting that Br-UTP incorporation in the PNC is due to transcription by RNA polymerases II and/or III. Treatment of cells with an RNA polymerase II and III inhibitor induces a significant reorganization of the PNC. In addition, double labeling experiments showed that poly(A) RNA and some of the factors required for pre-mRNA processing were localized in the PNC in addition to being distributed in their previously characterized nucleoplasmic domains. Fluorescence recovery after photobleaching (FRAP) analysis revealed a rapid turnover of polypyrimidine tract binding protein within the PNC, demonstrating the dynamic nature of the structure. Together, these findings suggest that the PNC is a functional compartment involved in RNA metabolism in the cell nucleus.

In situ analysis of changes in telomere size during replicative aging and cell transformation.

Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual decrease in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.

Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.

The intranuclear distribution of nuclear matrix-associated protein p107 and the 28-kD Sm antigen of U-snRNPs have been studied using double-label immunofluorescence and immunoperoxidase electron microscopy. In interphase nuclei of HeLa cells, Novikoff hepatoma cells, and rat kangaroo kidney cells, p107 was confined to discrete interchromatin domains. The domains had an irregular contour, with an average diameter of 1-1.5 micron. Each domain appeared to be composed of interconnected granules. The Sm antigen colocalized and appeared concentrated in these domains but also showed some general nucleoplasmic distribution. During mitosis, the interchromatin domains disassembled such that the Sm portion redistributed to the perichromosomal and spindle regions and the p107 component redistributed throughout the mitotic cytoplasm. During anaphase, p107 assembled into discrete clusters throughout the mitotic cytoplasm. The Sm antigen was not a component of these clusters. Double-label immunofluorescence with anti-p107 and the anti-DNA tight-binding protein, AhNa1, showed that the extranuclear p107 domains assumed an interchromatin localization only after the chromosomes had decondensed. The correlation between chromosome decondensation and the occurrence of p107 within interchromatin domains was also observed during chicken erythrocyte nuclear reactivation. We propose that the discrete interchromatin domains that contain p107 and p28 may be important for processing and splicing of RNA and that their structural assembly within nuclei is sensitive to the presence of the transcriptionally active conformation of chromatin.

Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo.

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors.

We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo.

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.