Expression Of Sorcin Research Articles

REIC/Dkk-3 stable transfection reduces the malignant phenotype of mouse prostate cancer RM9 cells

The reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3, a member of the Dkk gene family, is a tumor suppressor in a broad range of cancers. REIC/Dkk-3 transfected stable clones of mouse prostate cancer RM9 cells (RM9-REIC) and the empty vector-transfected control clone cells (RM9-EV) were established. Clones were used to evaluate the anti-cancer effects and a proteomics analysis of REIC/Dkk-3 continuous expression was performed. The RM9-REIC cells show a feeble appearance and the cell membrane shows irregular buds known as blebs. In vitro cell proliferation was significantly suppressed in RM9-REIC clones in comparison to the control. The apoptosis assay was done under standard culture conditions and RM9-REIC showed a higher incidence of apoptosis. The RM9-EV and RM9-REIC cells were orthotopically implanted into a C57BL/6 mouse prostate. After 2 weeks, the tumor growth was significantly inhibited in RM9-REIC cells in comparison to the control. Two-dimensional gel electrophoresis was used to examine the modification of protein expression by the gene transfection. The analysis with mass spectrometry disclosed that expression of peroxiredoxin-1, GST-P1, transgelin-2, MRP-L12, ARD, GRP78 and Sorcin were increased and eEF1A-1 and cyclophilin-40 protein were decreased in RM9-REIC cells. Therefore, REIC/Dkk-3 stable transfectants show a reduction of malignancy in mouse prostate cancer RM9 cells in vitro and in vivo. The result of the proteomics analysis might provide important clues to clarify the anti-cancer molecular mechanism of REIC/Dkk-3 gene transfer.

Ca2+‐Binding Proteins in Dogs with Heart Failure: Effects of Cardiac Contractility Modulation Electrical Signals

In dogs with heart failure (HF), chronic therapy with cardiac contractility modulation (CCM) electrical signals delivered to left ventricular (LV) muscle during the absolute refractory period improves LV function. This study examined the effects of CCM therapy on the expression of calcium (Ca(2+))-binding proteins (CBPs) in dogs with HF. Studies were performed in LV tissue from seven CCM-treated HF dogs, seven untreated HF dogs, and six normal (NL) dogs. mRNA expression of S100A1, sorcin, presenillin-1 (PS1), PS2, histidine-rich Ca(2+)-binding protein (HRC), and 18S ribosomal RNA (18S), a housekeeping gene, was measured using RT-PCR. Protein levels of CBPs and calsequestrin (CSQ) were determined by Western blotting. No difference was observed in the expression of 18S and CSQ among study groups. Compared with NL, the expression of S100A1, sorcin, and HRC was decreased, whereas the expression of PS2 was increased in untreated HF dogs. CCM therapy normalized the expression of S100A1, sorcin, and PS2 but not of HRC. No change was seen in the expression of PS1 among study groups. CCM therapy restores LV expression of S100A1, PS2, and sorcin. Normalization of CBPs may partly contribute to improved LV function in HF following CCM therapy.

Variation of Sorcin's Expression in the Reversion of Multidrug Resistance of K562/A02 Leukemic Line with Tetrandrine.

Objective This paper aims to investigate the reversal multiple of daunomycin(DNR) with different concentration of tetrandrine(tet) at different time, to study the variation of Soluble resistance related calcium binding protein (sorcin)’s expression in the reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tet at different time, and to provide new theoretic evidence for the clinical application of tet as resistence modifying agents. Method The reversal multiple of DNR with different concentration of tet at different time was assayed by MTT (concentration of tet is 0.5mg/l or1mg/l or 2mg/l, incubation time is 48h or 72h). The variation of the gene’s expression of sorcin with different concentration of tet at different time wasassayed by RT-PCR(grouping like MTT). Result MTT analysis demonstrated that the reversal concentration was 1.81(0.5mg/l,48h),3.62(1.0mg/l,48h),6.14(2.0mg/l,48h),2.8(0.5mg/l,72h),(1.0mg/l,48h),7.12(2.0mg/l,72h) respectively. RT-PCR analysis demonstrated that sorcin gene was lowly displayed in K562 cells and highly displayed in K562/A02 cells. The variation was first accentuation and then attenuation with the concentration accrescence of tet, this tendency had no obviously difference between 48h and 72h. Conclusion 1 The result of the experiment shows that tet may reverse multidrug resistance of K562/A02 cells by the down regulation of the expression of sorcin gene and proteinum; 2. It may have the concentration dependence and the time dependence in the reversion of multidrug resistance of K562/A02 cells with tet.

Sorcin modulation of Ca2+ sparks in rat vascular smooth muscle cells

Spontaneous, local Ca(2+) release events or Ca(2+) sparks by ryanodine receptors (RyRs) are important determinants of vascular tone and arteriolar resistance, but the mechanisms that modulate their properties in smooth muscle are poorly understood. Sorcin, a Ca(2+)-binding protein that associates with cardiac RyRs and quickly stops Ca(2+) release in the heart, provides a potential mechanism to modulate Ca(2+) sparks in vascular smooth muscle, but little is known about the functional role of sorcin in this tissue. In this work, we characterized the expression and intracellular location of sorcin in aorta and cerebral artery and gained mechanistic insights into its functional role as a modulator of Ca(2+) sparks. Sorcin is present in endothelial and smooth muscle cells, as assessed by immunocytochemical and Western blot analyses. Smooth muscle sorcin translocates from cytosolic to membranous compartments in a Ca(2+)-dependent manner and associates with RyRs, as shown by coimmunoprecipitation and immunostaining experiments. Ca(2+) sparks recorded in saponin-permeabilized vascular myocytes have increased frequency, duration and spatial spread but reduced amplitude with respect to Ca(2+) sparks in intact cells, suggesting that permeabilization disrupts the normal organization of RyRs and releases diffusible substances that control Ca(2+) spark properties. Perfusion of 2 mum sorcin onto permeabilized myocytes reduced the amplitude, duration and spatial spread of Ca(2+) sparks, demonstrating that sorcin effectively regulates Ca(2+) signalling in vascular smooth muscle. Together with a dense distribution in the perimeter of the cell along a pool of RyRs, these properties make sorcin a viable candidate to modulate vascular tone in smooth muscle.

Sorcin Regulates Excitation-Contraction Coupling in the Heart

Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart.

Expression of sorcin predicts poor outcome in acute myeloid leukemia

Using a cDNA microarray 12 differentially expressed genes were identified in a multidrug resistant (MDR) cell line K562/A02. The differential expression of sorcin, which was one of the 12 genes, has been confirmed by Northern blot. To determine the clinical role of sorcin, we have measured its expression in leukemic blast cells of 65 acute myeloid leukemia (AML) patients by reverse transcriptase polymerase chain reaction (RT-PCR). Sorcin overexpression in AML patients was associated with poor clinical outcomes, the complete remission (CR) rate in sorcin− cases was significantly higher than that of sorcin+ cases ( P<0.001). Furthermore, sorcin expression in AML patients was positively correlated with mdr1 expression ( r=0.841, P<0.001). Combination of sorcin and mdr1 was related to clinical outcome too, cases with sorcin−/mdr1− had best response to induction chemotherapy. Our results indicated that sorcin might be one of the factors that contributes to drug resistance of AML patients.

Transgenic Mice with Cardiac-Specific Expression of Activating Transcription Factor 3, a Stress-Inducible Gene, Have Conduction Abnormalities and Contractile Dysfunction

Activating transcription factor 3 (ATF3) is a member of the CREB/ATF family of transcription factors. Previously, we demonstrated that the expression of the ATF3 gene is induced by many stress signals. In this report, we demonstrate that expression of ATF3 is induced by cardiac ischemia coupled with reperfusion (ischemia-reperfusion) in both cultured cells and an animal model. Transgenic mice expressing ATF3 under the control of the alpha-myosin heavy chain promoter have atrial enlargement, and atrial and ventricular hypertrophy. Microscopic examination showed myocyte degeneration and fibrosis. Functionally, the transgenic heart has reduced contractility and aberrant conduction. Interestingly, expression of sorcin, a gene whose product inhibits the release of calcium from sarcoplasmic reticulum, is increased in these transgenic hearts. Taken together, our results indicate that expression of ATF3, a stress-inducible gene, in the heart leads to altered gene expression and impaired cardiac function.

Purification, cDNA Cloning, and Expression of Human Sorcin in Vincristine-Resistant HOB1 Lymphoma Cell Lines

Human sorcin,Mr22-kDa/pI5.3, was identified in HOB1 lymphoma cells resistant to 1.0 μMvincristine (designated HOB1/VCR1.0). The protein was purified and polyclonal antibody against it was produced. Using antibody to precipitate sorcin from32P-labeled cell extract, the SDS–PAGE-resolved human sorcin was not phosphorylated in contrast to the report of A. M. van der Blieket al.((1986)EMBO J.5, 3201–3208) in which the authors emphasized hamster sorcin might be phosphorylated through a cAMP-dependent protein kinase. Although sorcin had not been found among proteins of parental HOB1 cells labeled by [35S]methionine and resolved by 2-D PAGE, it could be immunoprecipitated from the cytosolic extract of the same cells.In vitrophosphorylation study did not reveal an enhanced phosphoprotein located in the area of 22 kDa, either. A 521-bp human sorcin cDNA fragment synthesized by PCR was used to screen cDNA library constructed from mRNA of HOB1/VCR1.0 cells. Human sorcin cDNA encoded 198 amino acids, 10 among which have been replaced in hamster sorcin. Of the 10 amino acid changes, 6 involve serine's in human sorcin compared to hamster's 1. These data suggest a difference in phosphorylation state between the two species. The results of Southern blot indicate the sorcin gene was amplified in HOB1/VCR1.0 cells under the pressure of high concentration of vincristine but not secondary to themdrgene amplification. 2-step PCR, exhibiting the gene could be latently expressed in parental HOB1 cells, confirms the above result of immunoprecipitation. Interestingly, HOB1/VCR0.5 cells (resistant to 0.5 μMvincristine) do not show amplification of the gene but do have increased expression of the protein. When this cell line was cultured in 0.1 μMvincristine for a period of time, the protein's expression amount reverted to the latent level. These results suggest that sorcin should have a place in mediating resistance of HOB1 cells to vincristine.

Association of Sorcin With the Cardiac Ryanodine Receptor

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.