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PubMed2005

Myocardial viability assessment by PET: (82)Rb defect washout does not predict the results of metabolic-perfusion mismatch.

UNLABELLED: PET is a sensitive technique for the identification of viable myocardial tissue in patients with coronary disease. Metabolic assessment with (18)F-FDG is considered the gold standard for assessment of viability before surgical revascularization. Prior research has suggested that viability may be assessed with washout of (82)Rb between early and late resting images. Our objective was to determine whether assessment of myocardial viability with (82)Rb washout is reliable when compared with PET using (18)F-FDG. METHODS: We performed PET for 194 patients referred for PET (18)F-FDG/(82)Rb to assess viability for clinical indications. We included 151 patients with resting defects >10% of the left ventricle (LV) (n = 159 defects). Patients with smaller resting (82)Rb defects (<10% LV) were excluded for the purpose of this study. PET images acquired with (82)Rb and (18)F-FDG defined viability by the mismatch between metabolism and perfusion ((18)F-FDG >125% of (82)Rb uptake in the (82)Rb defect). Evidence of viability with (82)Rb was assessed by the presence of (i) severity: (82)Rb counts in the defect >50% of (82)Rb in the normal zone of the resting PET images; (ii) washout: decrease of (82)Rb counts in the defect from early to late resting (82)Rb images <17% between the first 90-s image and the final 300-s image; or (iii) combined severity and washout criteria, which required positive criteria for (i) and (ii) to indicate viability. RESULTS: Prevalence of viability by (18)F-FDG/(82)Rb criteria was 50% (n = 79). Severity criteria yielded a sensitivity of 76% and a specificity of 17%, washout criteria yielded a sensitivity of 81% and a specificity of 23%, and both criteria had a sensitivity of 63% and a specificity of 32%. Positive and negative predictive values were poor for all criteria. No correlation existed between (82)Rb washout and (18)F-FDG-(82)Rb mismatch (r(2) = 0.00). Multiple receiver-operating-characteristic plots showed very poor discrimination despite varying criteria for viability by (82)Rb (severity from 50% to 60% of normal zone, washout from 12% to 17%). CONCLUSION: (82)Rb washout from early to late resting images, combined with quantitative severity of the resting (82)Rb defect, did not yield results equivalent to PET (18)F-FDG-(82)Rb mismatch and may not accurately assess myocardial viability.

Female↗
PubMedDate not supplied

Overexpression of c-fos induces expression of the retinoblastoma tumor suppressor gene Rb in transfected cells.

Hypophosphorylated Rb, the product of the tumor suppressor gene associated with hereditary retinoblastoma, is thought to act as a suppressor of cell growth and proliferation during G1 phase. We investigated whether Rb expression was dependent on the expression level of the immediate early cell growth gene, c-fos, which is transiently expressed as cells re-enter G1 phase from quiescence. To explore the functional relationship between c-fos and Rb, a eukaryotic expression plasmid was constructed containing the c-fos gene under control of the SV40 promoter complex. This plasmid was co-transfected with plasmids encoding pRSV cat and G418 resistance, into HeLa S3 cells, and clonal populations of transfected (RSfos) cells selected. High levels of c-fos expression in transfected cells were confirmed by both western and northern blot. Rb protein content per cell was determined by flow cytometry using an Rb-specific primary antibody and a FITC-conjugated secondary antibody. Higher expression of Rb (2-6 fold/cell) in approximately 20% of RSfos transfected cells was observed in comparison to parental HeLa cells. Rb content per cell increased approximately 2-fold during the cell cycle in both parental HeLa cells and RSfos cell clones which overexpressed Rb. Rb accumulation occurred in a manner consistent with normal mass accumulation during the cell cycle. Overexpression of Rb in RSfos cells was also confirmed by western blot analysis. Because one possible function of RB may be to act as a brake on cell growth, it is possible that overexpression of RB acts as an inhibitory counter activity to overexpression of the growth promoting activity of c-fos. This possible balancing activity of Rb was further suggested when Rb protein expression levels were measured in different clonal lines of RSfos transfected cells overexpressing increasing levels of c-fos. Overexpression of Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb expression may be regulated in a manner which balances the transcription stimulatory effects of c-fos overexpression and its effects on the transcription of other genes during the cell cycle.

Antibodies↗
PubMed1996

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro.

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.

Animals↗
PubMed1987

Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.

This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains.

Adenosine Triphosphate↗
PubMed1997

RB 101, a purported pro drug inhibitor of enkephalin metabolism, is antinociceptive in pregnant mice.

In an earlier study, we demonstrated the enhancement of pregnancy-induced analgesia with an inhibitor of endogenous enkephalin metabolism. The purpose of the present study was to evaluate the antinociceptive effect of another inhibitor of enkephalin metabolism, RB 101, on pregnant mice. Further, since other studies have shown RB 101 to be free of opioid side effects, we examined its effect on respiratory rate. Analgesia was assessed using the hot plate test, and respiratory rate was measured by recording the output from an end-tidal carbon dioxide detector. In pregnant mice, experiments were conducted on Day 17 or Day 18 of pregnancy; mice usually deliver on Day 19. For the hot plate test, animals were tested in the following groups: Group 1, RB 101 150 mg/kg (n = 15); Group 2, RB 101 50 mg/kg (n = 15); Group 3, RB 101 vehicle (n = 15); Group 4, morphine 5 mg/kg (n = 14); and Group 5, RB 101 150 mg/kg + naloxone 5 mg/kg (n = 10). The test was repeated on the second day after delivery in animals in Groups 1 and 3 (given RB 101 150 mg/kg and RB 101 vehicle, respectively). RB 101 150 mg/kg and morphine 5 mg/kg were significantly different (mean percentage of maximum possible effect 30.0 and 37.7, respectively, at 30 min and 41.6 and 32.6, respectively, at 60 min) in their antinociceptive effect in pregnant animals from all other groups. Naloxone, when coadministered with RB 101, prevented the development of antinociception. RB 101 150 mg/kg was not antinociceptive after delivery. Depression of respiratory rate was tested in a separate set of animals in the following groups: Group 1, RB 101 150 mg/kg (n = 16); Group 2, morphine 5 mg/kg (n = 16); Group 3, RB 101 vehicle (n = 15). Morphine 5 mg/kg produced significant depression of respiratory rate at 30 min postinjection when compared with RB 101 150 mg/kg and RB 101 vehicle (mean percent change in respiratory rate was 78.5% compared with 87.7% and 92.4%, respectively, where 100% = no change). These results suggest that drugs such as RB 101 may produce antinociception with minimal effects on respiration.

Analgesics↗
PubMed2000

Establishment of irreversible growth arrest in myogenic differentiation requires the RB LXCXE-binding function.

The crystal structure of the A-B domain of RB has defined the binding pocket for the LXCXE peptide motif. Using the crystal structure as a guide, we have inactivated the LXCXE-binding pocket by replacing N757 with Phe [to obtain RB(N757F)]. RB(N757F) does not bind to viral oncoproteins but retains the ability to bind and inhibit E2F. RB(N757F) is less effective than the wild-type RB [RB(WT)] in repressing E2F-regulated transcription, and its repression activity is not affected by trichostatin A, an inhibitor of histone deacetylases. However, RB(N757F) is as effective as RB(WT) in suppressing cell growth. Interestingly, RB(N757F) cannot establish an irreversible growth arrest in differentiated myocytes. Differentiated myocytes with RB(WT) become refractory to serum. By contrast, differentiated myocytes with RB(N757F) undergo DNA synthesis and phosphorylate RB(N757F) in response to serum, despite a high level of p21Cip1 expression. Mutation of the phosphorylation sites in RB(N757F) rescued its defect and allowed myocytes to permanently withdraw from the cell cycle. These results demonstrate that it is possible to inactivate the LXCXE-binding pocket without compromising the overall integrity of RB. Moreover, the LXCXE-binding pocket is dispensable for the intrinsic growth suppression function of RB. However, the LXCXE-binding function is essential for RB to establish the serum-refractory state in differentiated myocytes.

Amino Acid Motifs↗
PubMed1991

Loss of heterozygosity at the RB locus is frequent and correlates with muscle invasion in bladder carcinoma.

Studies of second, non-ocular tumours in surviving retinoblastoma patients and their families have reported a higher than expected incidence and lower age at diagnosis of bladder tumours. This suggests that RB mutations may predispose to bladder cancer. To determine whether this gene is involved in the development of sporadic bladder tumours we have examined 162 bladder tumours for evidence of structural alterations to the RB gene. Ninety-four patients were informative with one or more intragenic RB probes, and 28 of these (29%) showed loss of heterozygosity (LOH). Of these, two tumours showed homozygous deletions with the 5' intragenic probe p123M1.8. The probe p68RS2.0, which recognizes a variable number of tandem repeats site in intron 17 of the RB gene, detected new alleles in 5 of 162 tumours, one of which also showed LOH at another polymorphic site within the gene. The 28 tumours with RB LOH were screened with the RB cDNA probes pR3.8 and pR0.9, which revealed two homozygous deletions and one rearrangement. The tumours with RB LOH were also screened for loss of three markers which flank RB, D13S1 which maps proximal and D13S2 and D13S3 which map distal to RB on chromosome 13q. Two tumours showed retention of heterozygosity for flanking markers on one side of RB and another for markers on both sides. These results suggest that RB is the target gene on 13q in these bladder tumours. When RB loss was compared with tumour grade and stage, an association between high tumour grade and RB loss (0.005 greater than P greater than 0.001) and between muscle invasion and RB loss (P greater than 0.001) was found. Twenty-six of the 28 tumours with LOH were muscle-invasive. This represents 56% of invasive tumours. Only 2/48 (4%) superficial tumours showed RB allele loss, and one of these has progressed rapidly to invasive disease. These results show that LOH at the RB locus is a frequent genetic event in bladder tumours and may identify a subset of more aggressive tumours.

Adenocarcinoma↗
PubMed1993

Allosteric regulation of the access channels to the Rb+ occlusion sites of (Na+ + K+)-ATPase.

Previous work on the role of occluded Rb+ (a K+ substitute) in the reaction cycle of (Na+ + K+)-ATPase has focused on the kinetics of the dissociation of the enzyme-Rb+ complex at 20-24 degrees C. Doing experiments at 4 degrees C, we have made the following observations on the equilibrium binding levels and the kinetics of binding and release of Rb+. 1) The plot of bound Rb+ as a function of [Rb+] showed occupancy of high affinity sites, followed by binding to sites of lower affinity. The estimated number of Rb+ sites/active site was two to three, but a higher number was not ruled out. Release of bound Rb+ was slow and not monoexponential, the major portion being in a pool with a half-life of 4-5 h. Dissociation curves were identical at different levels of site occupancy. Rb+ binding also had fast and slow phases, requiring about 24 h to reach steady state at vastly different [Rb+]. These data suggest that (a) Rb+ occlusion sites are confined within the protein matrix and connected to the medium by narrow access channels that are heterogeneous in size, and (b) channel heterogeneity is distinct from differences in occlusion site affinities. 2) ATP, at a low affinity allosteric site, had no significant effect on the maximal level of bound Rb+ at any [Rb+], but it accelerated both the fast and the slow phases of Rb+ binding and release, and it increased the ratio of fast to slow phases. Evidently, ATP activates the channels (lowers the energy barrier for access) without altering binding site affinities. 3) Na+ was a competitive inhibitor of Rb+ at the occluded sites, but it also acted at an allosteric site to activate the access channels. Rb+ and K+ also had allosteric effects: although they did not affect the access channels directly, they blocked the allosteric effect of Na+. 4) Ouabain was an access channel inhibitor. It reduced the rates of binding and release of Rb+, blocked channel activation by ATP and Na+, but seemed to have no effect on the events at the occluded sites. The existence of heterogeneous access channels to the ion transport sites and the demonstration of channel regulation by the physiological ligands of the enzyme suggest the necessity of the inclusion of such allosteric mechanisms in the reaction cycle of (Na+ + K+)-ATPase.

Adenosine Triphosphate↗
PubMed2002

Active RB elicits late G1/S inhibition.

The retinoblastoma tumor suppressor protein (RB) is activated/dephosphorylated to mediate cell cycle inhibition in response to antimitogenic signals. To elucidate the mode of RB action at this critical transition, we utilized cell lines that can be induced to express a constitutively active allele of RB (PSM-RB). As expected, induction of PSM-RB, but not wild-type protein (WT), inhibited progression into S phase. It has been well documented that active RB inhibits E2F reporter activity, and this observation was confirmed upon induction of PSM-RB. Additionally, active RB inhibited E2F-2-mediated stimulation of cyclin E. By contrast, PSM-RB did not affect the mRNA or protein levels of endogenous cyclin E when mediating cell cycle inhibition. Similarly, there was no observable effect on cyclin E protein levels when p16ink4a was utilized to activate endogenous RB. CDK2/cyclin E complex formation was not disrupted and cyclin E-associated kinase activity was retained in the presence of PSM-RB. Additionally, centrosome duplication, a CDK2/cyclin E-dependent event, was not altered in the presence of active RB. Together, these data indicate that active RB does not block the G1/S transition through inhibition of cyclin E expression or activity. In contrast, PSM-RB leads to a dramatic reduction in cyclin A protein levels by coordinate transcriptional repression and degradation. This attenuation of cyclin A protein correlates with cell cycle inhibition. These studies indicate that RB inhibits cell cycle progression by targeting CDK2/cyclin A-dependent events at the G1/S transition to inhibit cell cycle progression.

Animals↗
PubMed2004

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.

The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.

Aneuploidy↗
PubMed1994

Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells.

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)-free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage-dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.

Animals↗
PubMed1999

pH regulation of K(+) efflux from myocytes in isolated rat hearts: (87)Rb, (7)Li, and (31)P NMR studies.

This study investigates the effects of intracellular (pH(i)) and extracellular pH (pH(e)) on the efflux of Rb(+) and Li(+) in isolated rat hearts. (87)Rb and (7)Li NMR were used to measure Rb(+) and Li(+) content, respectively, of hearts, and (31)P NMR was used to monitor pH(i), pH(e), and phosphate levels. After 30-min equilibration with Rb(+) or Li(+), effluxes were initiated by switching perfusion to a Rb(+)- or Li(+)-free, high-K(+) (20.7 mM) Krebs-Henseleit buffer with 15 microM bumetanide. Monensin (2 microM) increased pH(i) from 7.10 +/- 0.05 to 7.32 +/- 0.07 and resulted in activation of Rb(+) efflux; the first-order rate constant (k x 10(3), in min(-1)) increased from 42 +/- 2 to 116 +/- 16. Glibenclamide (4 microM) did not inhibit monensin-activated Rb(+) efflux (k = 110 +/- 17), whereas quinine (0.2 mM) slightly inhibited it by 19 +/- 9%. Infusion of 15 mM NH(4)Cl during Rb(+) washout increased k for Rb(+) efflux by 93% (81 +/- 8), which was glibenclamide and quinine insensitive, and caused a transient increase in pH(i) to 7.25 +/- 0.08. Intracellular Li(+) inhibited NH(4)Cl-stimulated Rb(+) efflux by 55%. Monensin and NH(4)Cl stimulated Li(+) efflux by 40%, increasing k from 29 +/- 3 to 43 +/- 7 and 41 +/- 3, respectively. The stimulation was not sensitive to 10 microM dimethylamiloride. Intracellular acidosis that resulted from the washout of NH(4)Cl (pH 6.86 +/- 0.2) slightly inhibited Rb(+) efflux (k = 36 +/- 5), whereas NH(4)Cl itself in the absence of pH(i) changes did not markedly affect Rb(+) efflux. A moderate increase in pH(i) (7.17 +/- 0.06) produced by washout of 15 mM 2, 2-dimethylpropionate (DMP)-Tris from hearts preequilibrated with DMP did not markedly affect Rb(+) efflux. Neither global alkalosis (pH(i) 7.4, pH(e) 7.55) nor acidosis (pH(i) approximately pH(e) 6.8) produced by 3 mM Tris base or 5 mM MES, respectively, affected Rb(+) efflux. We suggest that intracellular alkalosis stimulates Rb(+) (K(+)) and Li(+) effluxes by activating a nonselective sarcolemmal K(+) (Li(+))/cation exchanger or a K(+) (Li(+))-anion symporter.

Acidosis↗
PubMed1995

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study.

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.

Animals↗
PubMed1999

Loss of heterozygosity of the Rb gene correlates with pRb protein expression and associates with p53 alteration in human esophageal cancer.

To understand the alterations of Rb tumor suppressor gene and the relationship between defects in the Rb and p53 pathways in human esophageal carcinogenesis, we examined the loss of heterozygosity (LOH) of the Rb gene and immunohistochemical staining of pRb protein in 56 esophageal squamous cell carcinoma specimens and related the results to the p53 gene alterations. Using four introgenic polymorphic markers as probes, we observed LOH of the Rb gene in 30 of the 55 informative tumor samples. Immunohistochemical analysis revealed different patterns of pRb expression among the tumor samples. In the 56 cases, 16 displayed extensive pRb staining comparable to that of the adjacent normal epithelia, whereas 33 showed either significantly decreased or no pRb staining and 7 had a focal staining pattern reflecting heterogeneous cancer nests in the tumor with respect to Rb status. In the tumor samples containing Rb LOH, 90% showed low or no pRb expression, whereas in samples without Rb LOH, only 20% had altered pRb expression. There was a strong association between LOH of the Rb gene and alteration of pRb expression in our samples (P < 0.0001), suggesting LOH is a main event leading to Rb inactivation. We found that Rb LOH was more frequent in tumors with p53 mutations (P < 0.05), which occurred in 31 of the 49 cases analyzed. When the status of Rb and p53 alterations was evaluated by the combined results of immunohistochemical and genetic analyses, we found that alteration of Rb and p53 had an even stronger association in our esophageal squamous cell carcinoma samples (P = 0.0015). Among the 51 cases in which both the Rb and p53 status were determined, 31 contained alterations in both genes, and only 5 and 6 cases were altered in only Rb and only p53, respectively. Our results suggest that defects in the Rb and p53 pathways and their potential synergistic effect in deregulating cell cycle and apoptosis are major mechanisms for esophageal carcinogenesis.

Alleles↗
PubMed2002

Unbiased analysis of RB-mediated transcriptional repression identifies novel targets and distinctions from E2F action.

The retinoblastoma tumor suppressor, RB, is thought to inhibit cell cycle progression through transcriptional repression. E2F-regulated genes have been viewed as presumptive targets of RB-mediated repression. However, we found that specific E2F targets were not regulated in a consistent manner by the action of a RB allele that is refractory to cyclin-dependent kinase/cyclin-mediated phosphorylation (PSM-RB) when compared with E2F2 overproduction. Therefore, we used Affymetrix GeneChips as an unbiased approach to identify RB targets. We found that expression of PSM-RB significantly attenuates >200 targets, the majority of which are involved in cell cycle control (DNA replication or G2-M), DNA repair, or transcription/chromatin structure. The observed repression was due to the action of RB and not merely a manifestation of altered cell cycle distribution. Additionally, the majority of RB repression targets were confirmed through the blockade of endogenous RB phosphorylation via p16ink4a overexpression. Thus, these results have utility in assigning RB pathway activation in more complex systems of cell cycle inhibition (e.g., mitogen withdrawal, senescence, or DNA damage checkpoint). As expected, a significant fraction of RB-repressed genes have promoters that are bound/regulated by E2F family members. However, targets were identified that are distinct from genes known to be stimulated by overexpression of specific E2F proteins. Moreover, the relative action of RB versus E2F2 overexpression on specific genes demonstrates that a simple opposition model does not explain the relative contribution of RB to gene regulation. Thus, this study provides the first unbiased description of RB-repressed genes, thereby delineating new aspects of RB-mediated transcriptional control and novel targets involved in diverse cellular processes.

Animals↗
PubMed1988

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups.

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).

Animals↗
PubMed1993

Retinoblastoma (Rb) gene product expression in lymphomas. Correlation with Ki67 growth fraction.

The retinoblastoma susceptibility gene (Rb) has been characterized as a tumour suppressor gene. Rb protein is involved in cell-cycle control, regulating gene transcription. The absence of Rb protein in inherited retinoblastoma has been proved to be the result of inactivation of both Rb alleles through mutation or deletion, according to the general model for suppressor genes. The frequent detection of Rb gene alterations in human tumours (retinoblastoma, osteosarcoma, bladder carcinoma, small-cell lung carcinoma) and the correlation with clinical outcome found in some tumours prompted us to study Rb gene expression in lymphoid tumours in an attempt to determine whether Rb gene expression is related to histological type and degree of aggressivity in human lymphomas. To establish normal levels of Rb protein, its expression was analysed in vitro on cytospin preparations from normal and pokeweed mitogen (PWM) or phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs), using a monoclonal antibody (PMG3-245). Rb protein expression in vivo was quantified using a computer analysis system (CAS) on frozen sections from reactive and neoplastic lymphoid tissue. As a control of tissue preservation, and to compare Rb expression and growth fraction, the tumours and cells were labelled simultaneously with the Ki67 monoclonal antibody. Normal and stimulated lymphocytes showed a gradual increase of Rb protein during progression of the cell cycle, with a peak in the M phase. G0-G1 cells had no detectable levels of Rb protein, suggesting that the Rb gene may act as a 'status quo' cellular growth fraction control mechanism. In reactive lymphoid tissue, Rb protein was mainly expressed in germinal centres (lymph nodes, tonsils) and cortical thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1999

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity.

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.

Adenoviridae↗