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PubMed1997

Analysis of the Rb gene and cyclin-dependent kinase 4 inhibitor genes (p16INK4 and p15INK4B) in human ovarian carcinoma cell lines.

In the present study, we analyzed human ovarian carcinoma cell lines for abnormalities in the tumor suppressor gene Rb (retinoblastoma) and in cyclin-dependent kinase 4 (CDK4) inhibitor genes (p16INK4 and p15INK4B) using molecular biology techniques. For the Rb gene, in all six cell lines (PA-1, Caov-3 and -4, OVCAR-3, SK-OV-3, and Kuramochi), Rb gene abnormality was not detected using Southern blotting. In the Caov-3 cell line transcripts were not detectable by either Northern blot or polymerase chain reaction. Sequence analysis of the entire coding region of the Rb gene revealed point mutations (AAC to GAC) resulting in codon 123 (Asn to Asp) changes in the Caov-4 cell line. In the PA-1 cell line both wild-type Rb and mutant-type Rb (codon 798: CGG to TGG) were expressed, and in the OVCAR-3 cell line both wild-type Rb and mutant-type Rb (codon 704: ATG to GTG) were expressed. In four of six human ovarian carcinoma cell lines Rb gene abnormality was detected. For the p16INK4 and p15INK4B genes, only the SK-OV-3 cell line had abnormalities. There was a gene rearrangement or minor deletion of the p16INK4 gene in the SK-OV-3 cell line, while the p15INK4B gene was deleted in this cell line. In the SK-OV-3 cell line no mRNAs of p16INK4 and p15INK4B were expressed. At the point of Rb gene inactivation, we can explain five cell lines of six: four cell lines had abnormalities in the Rb gene itself, which is another mechanism by which the Rb gene is inactivated, while one cell line (SK-OV-3) had abnormalities in CDK4 inhibitor genes, another of the inactivation mechanisms of the Rb gene. These data suggest that abnormalities of Rb and CDK4 inhibitor genes (p16INK4, p15INK4B) may be involved in human ovarian carcinogenesis.

Carcinoma↗
PubMed1997

RB and hbrm cooperate to repress the activation functions of E2F1.

Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. We show here that in transfected cells RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex in vivo. Furthermore, RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, our results point out an unexpected role of corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. Finally, we demonstrate that the domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity.

Amino Acid Sequence↗
PubMed1993

Overproduction of Rb protein after the G1/S boundary causes G2 arrest.

The Rb protein is known to exert its activity at decision points in the G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of rapid inducible gene amplification to conditionally overexpress Rb protein during G2 phase. A cell line expressing a temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing the Rb cDNA linked to the simian virus 40 origin of replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDNA, a frameshift mutation close to the beginning of the Rb coding region, and a single-amino-acid deletion in the E1A/T-Ag binding pocket, respectively. Numerous independent cell lines were isolated at the nonpermissive temperature; cell lines displaying a high level of episomal amplification of an intact Rb expression cassette following shiftdown to the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activated cell sorting (FACS) analysis revealed that cultures induced to overexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB-fs or rearranged pRB-wt and did not express Rb protein had normal cell cycle profiles. Double-label FACS analysis showed that cells overexpressing Rb or Rb-Dra proteins were uniformly accumulating in G2, whereas cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 phase.

Animals↗
PubMed1998

RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interaction with transcription factor AP-2.

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. We recently reported that inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers including E-cadherin. Here we show that both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 was demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibited its binding to both RB and AP-2. Taken together, our results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc.

3T3 Cells↗
PubMed2004

The RB protein family in retinal development and retinoblastoma: new insights from new mouse models.

The Rb gene was isolated almost 20 years ago, but fundamental questions regarding its role in retinal development and retinoblastoma remain. What is the normal function of RB protein in retinogenesis? What is the cell-of-origin of retinoblastoma? Why do retinoblastoma tumors have recurrent genetic lesions other than Rb inactivation? Why is retinoblastoma not induced by defects in cell cycle regulators other than Rb? Why is the retina so sensitive to Rb loss? Recently developed conditional Rb knockout models provide new insight into some of these issues. The data suggest that RB protein may not control the rate of progenitor division, but is critical for cell cycle exit when dividing retinal progenitors differentiate into postmitotic transition cells. This finding focuses attention on the ectopically dividing transition cell, rather than the progenitor, as the cell-of-origin. Cell-specific analyses in the RB-deficient retina reveal that ectopically dividing photoreceptors, bipolar and ganglion cells die, but amacrine, horizontal and Muller cells survive and stop dividing when they terminally differentiate. Rare amacrine transition cells escape cell cycle exit and generate tumors. These data suggest that post-Rb mutations are required to overcome growth arrest associated with terminal differentiation, rather than apoptosis as previously suggested. To explain why perturbing cell cycle regulators other than RB does not initiate retinoblastoma, we speculate that mutations in other components of the RB pathway perturb cell cycle arrest, but only RB loss triggers genome instability in retinal transition cells, which may be critical to facilitate post-Rb mutations necessary for transformation. Cell-specific differences in the effect of Rb loss on genome stability may contribute to the tremendous sensitivity of retinal transition cells to tumorigenesis. The new mouse models of retinoblastoma will be invaluable for testing these possibilities.

Animals↗
PubMed1988

Altered expression of the retinoblastoma (RB) gene in small-cell carcinoma of the lung.

Nine lung small-cell carcinoma (SCC) cell lines and 9 lung non-SCC cell lines were examined for structural changes of the retinoblastoma (RB) gene as well as its expression using a complementary DNA probe. The RB protein product was investigated using an anti-RB antibody which we produced. Although homozygosity or hemizygosity of the RB gene was suggested in 8 of 9 SCCs and one of 2 large cell carcinomas (LCCs) by Southern blot analysis using an RB cDNA probe and polymorphic DNA markers for chromosome 13, no obvious structural changes of the RB gene were detected in these 18 cell lines. However, RB transcripts were either markedly reduced in quantity or abnormal in length in 3 of 9 SCCs. The specific 115 kD protein was not immunoprecipitated by the anti-RB antibody in all 9 SCCs with either normal or abnormal size RB mRNA. Three of 4 adenocarcinomas (AdCs), all 3 squamous cell carcinomas, and one of 2 LCCs expressed normal size RB mRNA, and the 115 kD protein was immunoprecipitated by the anti-RB antibody. The 115 kD protein was also absent in one of 2 LCCs with shortened RB mRNA and in one of 4 AdCs with low level of RB mRNA expression. These results strongly suggest that inactivation of the RB gene might be involved in the development of lung cancers, especially of SCCs.

Adenocarcinoma↗
PubMed1984

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation.

Influx of the K+ analogue Rb+ was measured through the ouabain-sensitive Na+/K+ pump and the ouabain-insensitive "leak" pathways in Cl- or NO3- in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb+ influxes by the two pathways were of about equal magnitude in Cl- (13 and 10 mmoles/liter cells X hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells X hr). In Na+ media, a portion of the ouabain-insensitive "leak" flux of Rb+ was Cl(-)-dependent (Rb+Cl- transport) as NO3- replacement reduced Rb+ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb+Cl- transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb+ pump and ouabain-insensitive Rb+Cl- influx were lost. In contrast, the NEM-stimulated Rb+Cl- transport changed much less throughout this period, suggesting an entity operationally but not necessarily structurally distinct from the basal Rb+Cl- transport. Although the experimental variability precluded a full assessment of significant changes in the small Na+/K+ (Rb+) pump and Rb+Cl- fluxes in mature pig red cells kept for the same time period in vitro, Rb+ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na+/K+ pump and Cl(-)-dependent K+ fluxes in vivo.

Animals↗
PubMed2006

Distinct roles for RB loss on cell cycle control, cisplatin response, and immortalization in Schwann cells.

Schwann cells play a critical role in peripheral nerve function. Regulated proliferation of Schwann cells is an important facet of the response to nerve injury; however, aberrant proliferation can give rise to Schwann cell tumors such as malignant peripheral nerve sheath tumors (MPNST). These tumors exhibit a range of genetic lesions that include loss of the retinoblastoma tumor suppressor (RB) pathway. RB plays a critical role in the regulation of cellular proliferation and its loss is a common event in human cancers. Here, the specific action of RB loss on Schwann cell proliferation and response to therapeutic intervention was explored. In primary mouse Schwann cells, conditional RB loss led to increased levels of critical cell cycle regulatory gene products, yet provided only a modest influence on proliferation. However, RB-deficient Schwann cells efficiently bypassed the cell cycle inhibitory response to the chemotherapeutic agent cisplatin, which is used in the treatment of MPNST and other glial tumors. Surprisingly, RB loss did not facilitate Schwann cell immortalization; and RB-deficient cells actually were less prone to immortalization than cells containing RB. Furthermore, RB-deficient cells that ultimately re-entered the cell cycle had lost both Schwann cell morphology and markers. Since, RB loss is likely a late event in Schwann cell tumor progression, the action of acute RB loss in immortalized Schwann cells was investigated. In this context, loss of RB had a profound effect on expression of target genes and the response to cisplatin. Thus, the loss of RB in both primary and immortal Schwann cells disrupted the response to anti-mitogenic signals and has implications for therapeutic intervention.

Animals↗
PubMed1999

p27 and Rb are on overlapping pathways suppressing tumorigenesis in mice.

The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. We have been able to study this relationship by genetic means by examining the phenotype of Rb+/-p27-/- mice. Tumors arise from the intermediate lobe cells of the pituitary gland in p27-/- mice, as well as in Rb+/- mice after loss of the remaining wild-type allele of Rb. Using these mouse models, we examined the genetic interaction between Rb and p27. We found that the development of pituitary tumors in Rb+/- mice correlated with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 was an indirect consequence of tumor formation or a contributing factor to the development of this tumor, we analyzed the phenotype of Rb+/-p27-/- mice. We found that these mice developed pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that was more aggressive than the disease in either Rb+/- or p27-/- mice. Importantly, we detected both pituitary and thyroid tumors earlier in the Rb+/-p27-/- mice. We therefore propose that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals.

Aging↗
PubMed1998

Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.

Adenovirus E1A Proteins↗
PubMed2003

Rb and N-ras function together to control differentiation in the mouse.

The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote differentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb(-/-); N-ras(-/-) skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb(-/-); N-ras(-/-) muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb(-/-); N-ras(-/-), compared to Rb(-/-) myoblasts in culture. The improved differentiation of skeletal muscle in Rb(-/-); N-ras(-/-) embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.

Animals↗
PubMed2002

Rb localization and phosphorylation kinetics correlate with the cellular phenotype of cultured breast adenocarcinoma cells.

Retinoblastoma protein (Rb) expression has been correlated with state of differentiation, proliferation rate, and metastatic potential in breast adenocarcinomas and established cell lines. These observations, based on immunoreactivity of total Rb rather than hypophosphorylated protein, do not address the relationship between functional Rb and indicators of an aggressive transformed cellular phenotype. We hypothesized that the distribution of functional Rb and the kinetics of Rb phosphorylation would differ between cell lines representing immortalized mammary epithelium (MCF10A), differentiated nonmetastatic mammary adenocarcinoma (MCF-7), and poorly differentiated, highly metastatic mammary adenocarcinoma (MDA-MB-231) and that these differences would be informative of the cellular phenotype. Direct immunofluorescence microscopy was used to compare qualitatively the subcellular localization of total and hypophosphorylated Rb protein in synchronized and asynchronous cells. This technique was also used to quantitatively assess the amounts of hypophosphorylated Rb throughout the cell cycle in these representative cell lines. Total Rb stained more prominently than hypophosphorylated Rb in the nucleus of all asynchronous cells. Rb phosphorylation was more rapid in MCF-7 cells than in MCF10A cells, whereas Rb dephosphorylation appeared deregulated in MDA-MB-231 cells. We conclude that assessment of hypophosphorylated Rb may be more useful than assessment of total Rb for the evaluation of transformed breast adenocarcinoma phenotypes.

Adenocarcinoma↗
PubMed1995

The retinoblastoma gene (RB-1) status in multiple myeloma: a report on 35 cases.

We looked for abnormalities of the retinoblastoma (RB-1) gene and of RB protein expression in 35 patients with multiple myeloma (MM). Mutations in exons 20 to 24 of the RB-1 gene (exons where mutations predominate in retinoblastoma and other solid tumors) were analyzed by single stranded conformation polymorphism (SSCP). RB-1 protein was studied in bone marrow plasma cells by immunocytochemistry (ABC peroxidase technique) with a specific monoclonal antibody. Southern blot analysis of RB-1 gene was also performed in 20 of the patients. Twenty two patients analyzed had advanced disease (stage III or, in one case, plasma cell leukemia) and cytogenetic analysis (performed in 31 cases) found monosomy 13 in 9 patients. No rearrangement of the RB-1 gene was found by Southern analysis. Absent or greatly reduced RB-1 protein level was found in plasma cells in 4 of the patients (11%), whereas normal levels were seen in the remaining cases. No point mutation in exons 20 to 24 and their flanking introns were found in any of the 35 patients. Three of the 4 patients with absent or reduced RB-1 protein expression had advanced MM (stage III: 2 cases; plasma cell leukemia: 1 case); all 4 patients were resistant to treatment (as compared to 7 of the 31 patients with normal RB-1 protein levels); only one of them was subsequently found to have monosomy 13 (as compared to 9 of the 28 other karyotyped patients). Our findings suggest that abnormalities of the RB-1 gene and its expression are rare in MM. Absent or reduced expression of RB-1 protein was not significantly correlated to monosomy 13 and was not associated with gross rearrangements of the RB-1 gene by Southern analysis or point mutations in exons 20 to 24 of the gene. Reduced expression of RB-1 protein may be associated with advanced disease and poor response to treatment, although larger numbers of patients will be required for more adequate conclusions.

Base Sequence↗
PubMed1998

Expression of the retinoblastoma tumor suppressor gene (RB-1) in acute leukemia.

In this report we review current studies concerning the RB-1 gene expression in acute leukemias. The RB-1 gene was analyzed in several studies by protein-, RNA and DNA-techniques in acute lymphoblastic leukemia (ALL) as well as in acute myelogenous leukemia (AML). The frequency of RB-1 inactivation in ALL-patients ranged between 30% and 64% in several studies. Structural abnormalities of the RB-1 gene were reported in 18% of ALL-patients and in 27% of Philadelphia chromosome-positive ALL, respectively. The proportion of AML-patients with absent RB-1 protein expression ranged between 19% and 55%. Structural RB-1-abnormalities in AML were predominantly reported in leukemias with monocytic differentiation. Furthermore, the prognostic value of an abnormal RB-1 gene expression was also estimated in some studies. In childhood ALL RB-1 inactivation was reported to have prognostic significance while in contrast, in another study on adults no prognostic value of RB-1 was found. In 4 out of 5 documented studies AML-patients with RB-1 inactivation generally had a poorer prognosis. In conclusion, RB-1 inactivation is frequently observed in acute leukemia. The prognostic value of low RB-1 expression is controversial but the majority of published studies found low RB-1 expression to be a negative prognostic predictor, in acute leukemia.

Adult↗
PubMed1989

Rubidium (Rb) treatment of rats: biological effects and implications for psychiatry.

Experiments were carried out on rats in order to find out the implications for psychiatry of the basic biological effects of rubidium (Rb). The forced swimming test (FST), used to evaluate the effects on rats of treatment with Rb, was conducted after 1 or 3 mEq/kg Rb was given chronically or subacutely. The weight of rats treated with 1 or 3 mEq/kg Rb, once daily, was observed daily for two weeks. Rb levels in the blood and brain of rats treated with Rb chronically or subacutely were determined by atomic absorption spectrophotometry. The rectal temperature was observed at 30, 60, 90, 120, 180, and 240 min after injections of 1 or 3 mEq/kg Rb. The increase in the mean body weight of treated rats was almost the same as that of the control. The rectal temperature in rats treated with Rb showed a hyperthermic response. With both the 1 and 3 mEq/kg Rb treatments, Rb levels in the brains were significantly higher in the chronic experiments than in the subacute experiments. The experiments conducted to determine the effect of chronic and subacute treatment of Rb are hereafter termed 'chronic experiment' and 'subacute experiment' respectively. Almost the same significant difference was also observed in the Rb levels in the blood. In the FST, a decrease in the mean immobility time was not observed in either the subacute or the chronic experiments. Thus, antidepressant effects, as judged from the FST, were not observed although Rb did actually accumulate in both brain and blood.

Animals↗
PubMed1985

Relationship between immune system and gram-negative bacteria. III. Functional analysis of human peripheral blood lymphocyte subpopulations separated by cytoadherence with Salmonella minnesota Rb.

Spontaneous adherence of bacteria to human peripheral blood mononuclear cells (PBMC) represents a useful tool for analysis of lymphocyte subsets with different functions. We have recently shown that PBMC can be divided into 2 populations based on their ability to bind Salmonella minnesota R345 (Rb) bacteria. By using these procedures, here, we provide evidence that Rb-bound and Rb-unbound PBMC populations give similar proliferative responses to phytohemagglutinin (PHA) and concanavalin A (Con A), while the pokeweed mitogen (PWM)-induced proliferative and differentiative responses are higher in the Rb-unbound than in the Rb-bound PBMC fraction. Moreover, enhanced PWM-induced responses are obtained in Rb-unbound cell cultures enriched for T4+ cells. When B (non-E rosetting) cells are cultured with purified T lymphocytes from the Rb-bound (T-Rb+) and Rb-unbound (T-Rb-) fractions, comparable PWM-induced mitogenic responses are observed. The T-Rb- population contains a higher percentage of cells expressing T4+ phenotype, and when added to B cell cultures a more elevated PWM-induced IgA, IgG and IgM synthesis is observed than in B cell cultures containing T-Rb+ cells. These results suggest that the T-Rb- fraction is enriched for T cells which help IgA, IgG and IgM responses.

B-Lymphocytes↗
PubMed2000

Effects of ischemia on intracellular rubidium in pig and rat hearts: (87)Rb NMR imaging and spectroscopic study.

87Rb MR imaging and spectroscopy were used to study the effects of ischemia on the properties of K(+) in cardiac tissue. Isolated pig and rat hearts perfused by the Langendorff method with Krebs-Henseleit buffer were preloaded with Rb(+). Ischemia (Isc) was induced by 120-min occlusion of the left anterior descending artery in the pig hearts or by stopping perfusion for 33 min in the rat hearts. Serial (87)Rb MR images or spectra from the anterior (An) LV wall of pig hearts were acquired continuously. The intensities of the Rb images of the An and posterior (Pos) walls were similar and stable during the first 45 min of ischemia. The intensity of signal from the An wall (Isc) then gradually increased by 60 +/- 8% relative to the preischemic value (vs. 31 +/- 5% increase in Pos wall) and necrosis (19 +/- 5% of the LV wall mass) developed upon reperfusion. The Rb(+) content was lower in the ischemic (An) than in the normal (Pos) area (22.3 +/- 3 vs. 28.4 +/- 1.3 mmol/g wet wt). A similar pattern was observed in the peak heights of (87)Rb spectra from the An wall, which increased by 40 +/- 16% (vs. 21 +/- 11% in control) due to a 12% decrease in the apparent Rb linewidth (LW) and a 24 +/- 14% increase in the peak area. The Rb peak comprised narrow (297 +/- 21 Hz) and broad (1098 +/- 40 Hz, 59 +/- 3% of total area) Lorentzian components. The LW of the broad component decreased by 14%, while the narrow component did not change markedly. In the rat hearts ischemia caused a 33 +/- 4% increase in the (87)Rb peak height as a result of peak narrowing (13 +/- 1%), and an increase in peak area (17 +/- 5%). The decreases in LW and increases in Rb(+) visibility can be explained by an increase in Rb(+) mobility caused by displacement of Rb(+) from anionic binding sites by H(+) (ischemic acidosis) and changes in intracellular compartmentalization of Rb(+). Magn Reson Med 44:193-200, 2000.

Analysis of Variance↗
PubMed1998

Stable expression of antisense Rb-1 RNA inhibits terminal differentiation of mouse myoblast C2 cells.

To determine the roles of the retinoblastoma gene (Rb-1) in skeletal muscle differentiation in vitro, we isolated C2 myoblasts stably expressing an antisense RNA directed to the 3'-untranslated region (3'UTR) of Rb-1 mRNA. The levels of Rb-1 mRNA and its product (pRb) in the clones transfected with antisense Rb were markedly decreased to 25-35% of those in the control clone. Cell growth of the clones was accelerated, especially in medium containing low concentrations of fetal calf serum. Even in differentiation medium with a low mitogen level, the antisense Rb clones proliferated as single-nucleated myoblast-like cells without expressing the sarcometric myosin heavy chain protein, whereas the control clone formed highly multinucleated myotubes after 4 days of culture under the same conditions. Under this condition, the levels of Rb-1 mRNA and pRb in the antisense Rb clones were 30-50% of those in the control clone, and no divergent increase in the Rb-family protein p107 expression was observed. This inhibited differentiation was abrogated by reintroducing expression vectors for the sense 3'UTR of Rb-1 mRNA or Rb-1 mRNA lacking its 3'UTR to the clone transfected with antisense Rb. In the antisense Rb clone cultured in differentiation medium, the amounts of MyoD and myogenin mRNA were markedly decreased on the 2nd day of culture in the differentiation medium. The expression of cell cycle-promoting genes including E2F-1 and cyclin D1 was up-regulated throughout the experiment. These results demonstrate that pRb is essential for the completion of terminal differentiation in C2 cells.

Actins↗