Human Leukemia Cell Line K562 Research Articles

Polygonumins A, a newly isolated compound from the stem of Polygonum minus Huds with potential medicinal activities

Polygonumins A, a new compound, was isolated from the stem of Polygonum minus. Based on NMR results, the compound’s structure is identical to that of vanicoside A, comprising four phenylpropanoid ester units and a sucrose unit. The structure differences were located at C-3″″′. The cytotoxic activity of polygonumins A was evaluated on several cancer cell lines by a cell viability assay using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The compound showed the highest antiproliferative (p < 0.05) activities against K562 (Human Leukaemia Cell Line), MCF7 (Human breast adenocarcinoma cell line), and HCT116 (Colorectal cancer cells) cells. Cytotoxic studies against V79–4 cells were carried out and showed that polygonumins A was toxic at 50 µg/ml, suggesting that this compound may be used as an anticancer drug without affecting normal cells. Polygonumins A also showed promising activity as an HIV-1 protease inhibitor with 56% relative inhibition. Molecular docking results indicated that the compound possesses high binding affinity towards the HIV protease over the low binding free energy range of -10.5 to -11.3 kcal/mol. P. minus is used in Malaysian traditional medicine for the treatment of tumour cells. This is the first report on the use of P. minus as an HIV-1 protease inhibitor.

The effect of DNA hydroxymethylase Tet2 on γ globin activation in the treatment of β-thalassemia

Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with β- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for β thalassemias.

Apoptotic Effects of Some Tetrahydronaphthalene Derivatives on K562 Human Chronic Myelogenous Leukemia Cell Line

Retinoids which are vitamin A (Retinol) derivatives have been suggested to mediate the inhibition of cancer cell growth and apoptosis. It has been reported that all trans retinoic acid (ATRA) exhibited suppressive effects on different types of leukemia including chronic myelogenous leukemia. In the present study, we aim to find out the effects of 6 synthetic N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalene-2-yl)-carboxamide derivatives (compound 6-12) on cell viability and apoptotic pathways in K562 human chronic myelogenous leukemia cell line. Cell viability and apoptosis were examined by spectrophotometric thiazolyl blue tetrazolium bromide (MTT) and caspase-3 assay, western blot, RT-PCR and flow cytometry. Our results indicated that compound 6 (5-(1,2-Dithiolan-3-yl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)pentanamide), 8 (4-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)butanamide) and 11 (E-3-(4-Hydroxy-3-methoxyphenyl)-N-(3,5,5,8,8-pentamethyl- 5,6,7,8-tetrahydronaphthalen-2-yl)acrylamide) exhibited apoptotic effects in K562 human chronic myelogenous leukemia cell line and induced caspase 3, PARP cleavage, Bax/Bcl-2 ratio, Bad and Bim gene expressions. Some retinoid derivatives tested in this study induced apoptosis of K562 cells which suggest that these compounds may serve as potential agents in the treatment of chronic myelogenous leukemia.

Theoretical and experimental investigation of anticancer activities of an acyclic and symmetrical compartmental Schiff base ligand and its Co(ii), Cu(ii) and Zn(ii) complexes.

A compartmental Schiff base ligand, 2,2′-((((((2-hydroxypropane-1,3-diyl)bis(oxy))bis(2,1-phenylene))bis(methylene))bis(azanylylidene))bis(methanylylidene))bis(4-bromophenol) (H3LBr) and its complexes with cobalt(ii), copper(ii) and zinc(ii) including, [Co(HLBr)] (1), [Cu2(LBr)(μ-1,3-OAc)]·MeOH (2) and [Zn(HLBr)] (3) were prepared using template synthesis and characterised by elemental analysis, FT-IR and 1H NMR spectroscopies and single-crystal X-ray diffraction. In the structure of complexes 1 and 3 the metal atom has a MN2O2 environment with tetrahedral geometry while complex 2 has a binuclear structure with a MNO4 environment and square planar geometry around the copper atom. The ability of all compounds to interact with the nine biomacromolecules (BRAF kinase, CatB, DNA gyrase, HDAC7, rHA, RNR, TrxR, TS and Top II) are investigated by docking calculations. For examination of the docking results, the in vitro activities of eight compounds against the human leukemia cell line K562 was investigated by evaluation of IC50 values and mode of cell death (apoptosis).

Tsavoenones A-C: unprecedented polyketides with a 1,7-dioxadispiro[4.0.4.4]tetradecane core from the lichen Parmotrema tsavoense.

New racemic dispiranic polyketides, tsavoenones A (1), B (2) and C (3), having a novel 1,7-dioxadispiro[4.0.4.4]tetradecane scaffold were isolated from the foliose lichen Parmotrema tsavoense. These compounds were structurally elucidated by extensive NMR analyses, comparison between experimental and theoretical 13C NMR data and X-ray crystallography. A putative biosynthetic scenario for the formation of 1-3 from parmosidone D, a meta-depsidone previously isolated from the same lichen material, was proposed. Tested for its cytotoxicity, 1 displayed a moderate activity against human myelogenous leukemia K562 cell line with an IC50 value of 66 μg mL-1.

Self-assembling nanoparticles based on cytarabine prodrug for enhanced leukemia treatment

Cytarabine (Ara-C) is an attractive chemotherapeutic agent used for the treatment of acute myeloblastic leukemia, however, its severely drawbacks such as low lipophilicity and rapid plasma degradation limit clinical applications. Here, we synthesized a new Ara-C prodrug DTA-Ara by conjugating 2-decyltetradecanoic acid (DTA), a double-chained fatty acid with 24 carbons with Ara-C. It was the first time to see that DTA-Ara molecules could self-assemble into stable spherical nanoparticles (NPs) in aqueous solution with extremely high drug loading (63wt%). The DTA-Ara NPs had the average sizes of approximately 130nm and a zeta potential around −31.6mV. Importantly, the DTA-Ara NPs were stable in deionized water or phosphate buffer solution (PBS, pH7.4) solution for more than one week and the hemolysis rate was <10%, which indicated that it could be administrated intravenously. Moreover, the in vitro cytotoxicity study further manifested that the resulting prodrug showed the marked antitumor activity against human leukemia cell line K562 and HL60 cells compared to the naked drug ascribed to its improvement of the hydrophobicity and biomembrane penetrability. The strategy of delivering lipophilized nucleoside analogs using prodrug self-assembling nanoparticles demonstrate potential superiority for Ara-C and provide a new promising therapeutic schedule for leukemia.

Immune function in arctic mammals: Natural killer (NK) cell-like activity in polar bear, muskox and reindeer

Natural killer (NK) cells are a vital part of the rapid and non-specific immune defense against invading pathogens and tumor cells. This study evaluated NK cell-like activity by flow cytometry for the first time in three ecologically and culturally important Arctic mammal species: polar bear (Ursus maritimus), muskox (Ovibos moschatus) and reindeer (Rangifer tarandus). NK cell-like activity for all three species was most effective against the mouse lymphoma cell line YAC-1, compared to the human leukemia cell line K562; NK cell response displayed the characteristic increase in cytotoxic activity when the effector:target cell ratio increased. Comparing NK activity between fresh and cryopreserved mouse lymphocytes revealed little to no difference in function, highlighting the applicability of cryopreserving cells in field studies. The evaluation of this important innate immune function in Arctic mammals can contribute to future population health assessments, especially as pollution-induced suppression of immune function may increase infectious disease susceptibility.

Three New Cytotoxic Steroidal Glycosides Isolated from Conus pulicarius Collected in Kosrae, Micronesia.

Three new sulfated steroidal glycosides (3–5), along with known cholesterol derivatives (1,2), were isolated from the visceral extract of the cone snail Conus pulicarius. The structure of each new compound was elucidated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The three new compounds exhibited significant in vitro cytotoxicity (GI50 values down to 0.49 μM) against the K562 human leukemia cell line.

Proteomic analysis of affinity-purified extracellular proteasomes reveals exclusively 20S complexes.

Proteasome-mediated proteolysis is important for many basic cellular processes. In addition to their functions in the cell, proteasomes have been found in physiological fluids of both healthy and diseased humans including cancer patients. Higher levels of these proteasomes are associated with higher cancer burden and stage. The etiology and functions of these proteasomes, referred to as circulating, plasmatic, or extracellular proteasomes (ex-PSs), are unclear. Here we show that human cancer cell lines, as well as human endometrium-derived mesenchymal stem cells (hMESCs), release proteasome complexes into culture medium (CM). To define ex-PS composition, we have affinity purified them from CM conditioned by human leukemia cell line K562. Using matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), we have identified core 20S proteasome subunits and a set of 15 proteasome-interacting proteins (PIPs), all previously described as exosome cargo proteins. Three of them, PPIase A, aldolase A, and transferrin, have never been reported as PIPs. The study provides compelling arguments that ex-PSs do not contain 19S or PA200 regulatory particles and are represented exclusively by the 20S complex.

Design, synthesis and biological evaluation of substituted aminopyridazin-3(2H)-ones as G0/G1-phase arresting agents with apoptosis-inducing activities

A series of aminopyridazin-3(2H)-one derivatives has been designed and synthesized. Their antiproliferative activities were evaluated against three human cancer cell lines (SH-SY5Y human neuroblastoma, K562 human myelogenous leukemia and AGS gastric cancer cell lines) using the MTT assay. The preliminary activity test displayed that compound 8a exhibited comparable activities against all test cells with the positive control fluorouracil. Meanwhile compounds 8b, 8e and 9c-e displayed selective antiproliferative activities for SH-SY5Y cells. Furthermore, compounds 8a-b with low-micromole GI50 value for SH-SY5Y cells induced apoptosis with cell cycle arrest at G0/G1 phase in SH-SY5Y cells in a dose-dependent manner.

Dose-rate effect of ultrashort electron beam radiation on DNA damage and repair in vitro.

Laser-generated electron beams are distinguished from conventional accelerated particles by ultrashort beam pulses in the femtoseconds to picoseconds duration range, and their application may elucidate primary radiobiological effects. The aim of the present study was to determine the dose-rate effect of laser-generated ultrashort pulses of 4 MeV electron beam radiation on DNA damage and repair in human cells. The dose rate was increased via changing the pulse repetition frequency, without increasing the electron energy. The human chronic myeloid leukemia K-562 cell line was used to estimate the DNA damage and repair after irradiation, via the comet assay. A distribution analysis of the DNA damage was performed. The same mean level of initial DNA damages was observed at low (3.6 Gy/min) and high (36 Gy/min) dose-rate irradiation. In the case of low-dose-rate irradiation, the detected DNA damages were completely repairable, whereas the high-dose-rate irradiation demonstrated a lower level of reparability. The distribution analysis of initial DNA damages after high-dose-rate irradiation revealed a shift towards higher amounts of damage and a broadening in distribution. Thus, increasing the dose rate via changing the pulse frequency of ultrafast electrons leads to an increase in the complexity of DNA damages, with a consequent decrease in their reparability. Since the application of an ultrashort pulsed electron beam permits us to describe the primary radiobiological effects, it can be assumed that the observed dose-rate effect on DNA damage/repair is mainly caused by primary lesions appearing at the moment of irradiation.

Polyphyllin D inhibits proliferation of K562 on human leukemia cells by inducing apoptosis and differentiation

Objective To investigate the inhibitory effect of polyphyllin D on the proliferation of human chronic myelogenous leukemia (CML) cell line K562 cells and its mechanism. Methods Methyl thiazolyl thiazolium (MTT) assay was used to evaluate the inhibition effect of polyphyllin D on the proliferation of K562 cells. Flow cytometry was used to determine the effect of polyphyllin D on the apoptosis of K562 cells. The related proteins were analyzed by Western blotting. Results The MTT assay showed that polyphyllin D obviously inhibited the growth of K562 cells. The hemi-inhibitory concentration (IC50) of 24 hours for the K562 cells was (0.8±0.1)μmol/L. Flow cytometric results demonstrated that polyphyllin D significantly induced apoptosis in a dose-dependent manner, and the apoptosis rates of 0, 0.4, 0.8, 1.2 μmol/L polyphyllin D were (1.73±0.33)%, (14.47±2.57)%, (24.10±3.52)%, (31.51±4.09)% after 24 hours, with a significant difference (F=58.231, P<0.001). Western blotting showed that polyphyllin D significantly down-regulated the expression of Bcl-2, and this was accompanied by up-regulation of Bax, decrease of the Bcl-2/Bax ratio, up-regulation of cytochrome c and activation of caspase-3. Otherwise, polyphyllin D obviously induced monocyte differentiation by increasing CD14 expression on the surface of K562 cells. Conclusion Polyphyllin D can significantly inhibit the proliferation of K562 cells, and the mechanism may be realized by inducing apoptosis and promoting monocyte differentiation. Key words: Leukemia; Apoptosis; Cell differentiation; Polyphyllin D

ID1056 Synthesis and biological evaluation of novel curcumin derivative with water-soluble phosphate group as potential antitumor agent

Background: Curcumin (diferuloylmethane) is the main natural active polyphenol from the rhizome of Curcuma longa (turmeric) and in others Curcuma spp. The main pharmacological effects include anti-tumor, anti-inflammatory, anti-oxidation, anti-fungal and anti-bacterial activities. However, the clinical application of curcumin is limited by its main drawbacks such as low solubility, poor bioavailability and rapid excretion from the body. In this study, the chemical modification approach was performed to improve the solubility of curcumin in water, at the same time increase the anti-tumor activity.&#x0D; Methods: The water-soluble compound (2-(curcumin-O-yl)ethyl dihydrophosphate, LH-42017) was synthesized by two classical reactions: O-alkylation with 2-bromoethanol to obtain mono-O-(2-hydroxyethyl)-curcumin, followed by esterification using phosphoryl chloride in water and pyridine. The chemical structure was confirmed by IR-, NMR-spectroscopies and mass-spectrometry. The anti-tumor activity of LH-42017 was assessed against hepatocellular carcinoma Hep-G2, human breast cancer MCF-7, human leukemia K562 and human cervical carcinoma HeLa cell lines based on the sulforhodamine B (SRB) assay.&#x0D; Results: LH-42017 was successfully synthesized from curcumin by overall yield of 28.43%. IC50 of original curcumin was 63.55, 90.88, 104.99 and 60.24 µM on Hep-G2, MCF-7, K562 and Hela, respectively. IC50 of LH-42017 was 36.82, 51.60, 60.80 and 26.14 µM on Hep-G2, MCF-7, K562 and Hela, respectively.&#x0D; Conclusion: The novel synthesized compound LH-42017 showed the antitumor activity in 1.7-2.3 times better than original curcumin. The water-soluble phosphate group plays an important role in development of natural curcumin as potential antitumor agent.

Mechanisms of inhibiting human leukemia cell lines by serum of rats treated with compound banmao capsule.

Compound banmao capsule (CBC) is a traditional Chinese medicinal formula composed of extracts from 11 organisms. The present study investigated the mechanism of CBC on the biological behavior of human leukemia cell lines using seropharmacological methods. CBC-containing rat serum was prepared by intragastrical administration of CBC to rats. The proliferation of human leukemia HL60 and K562 cell lines was assayed by measuring cell viability with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium method, while cell cycle distribution and the rate of apoptosis were evaluated with flow cytometry. The mRNA expression of vascular endothelial growth factor A (VEGF-A) and chemotactic and inflammatory genes in human leukemia cell lines was examined using reverse transcription quantitative-polymerase chain reaction methods. It was revealed that the proliferation of K562 and HL60 cells was significantly inhibited by the CBC-containing rat serum at 72 h. The CBC-containing serum also promoted the apoptosis of K562 and HL60 cell lines. The CBC-containing serum altered the cell cycle progression of K562 and HL60, increasing the proportion of the cells in G1 phase and decreasing the proportion of the cells in S phase. Attenuated expression of VEGF-A and a decreasing trend in the expression of chemotactic and inflammatory genes were identified following treatment with CBC-containing serum in HL60 and K562 cells. In conclusion, CBC-containing serum exerted an inhibitory effect on the growth of K562 and HL60 cells by decreasing cellular proliferation, promoting apoptosis and cell cycle arrest, and decreasing the expression of VEGF-A, and chemotactic and inflammatory genes.

In vitro studies of the anticancer action of Tectaria cicutaria in human cancer cell lines: G0/G1 p53-associated cell cycle arrest-Part I

ObjectiveThe rhizome of Tectaria cicutaria has been used in Indian traditional medicine for the treatment of various disorders. The objective of present investigation is to screen various extracts of the rhizomes of Tectaria cicutaria for anti-cancer activity and to investigate the mechanism involved. Materials and methodsThe rhizomes of Tectaria cicutaria were extracted with different solvents. In vitro anti-cancer activity of different rhizome extracts were studied in Human cancer Cell Lines using Sulphorodamine B (SRB) colorimetric cytotoxicity assay. The effect of ethanolic extract (TCe) on cell growth inhibition, modulation in gene expression, and induction of apoptosis using the K562 human leukemia cell line were studied. The extract was analyzed by GC-MS to identify their major chemical compounds. ResultsTCe shows antioxidant potential in both DPPH scavenging assay and reducing capacity. Flow cytometric analysis showed that 11 μg/ml of TCe arrested cell cycle progression at the G0/G1 phase. In the TCe treated K562 cells, the mRNA and protein expression level of p53 was strongly up-regulated in reverse transcription polymerase chain reaction. Furthermore, its downstream target p21 level was also increased. The GC-MS study has depicted results with the presence of twelve different compounds which will require significant further efforts for structure and putative identification. ConclusionThe present work has for the first time, tried to elucidate the anti leukemic potential of Tectaria cicutaria. TCe was more potent in K562 cells, altering the cell cycle progression and inducing apoptosis.

Synthesis, crystal structure, and biological evaluation of ethyl 9-(2-fluorobenzyl)-1-phenyl-9 h-pyrido[3,4-b]indole-3-carboxylate as anticancer agent

ABSTRACTAn efficient method was described for the synthesis of ethyl 9-(2-fluorobenzyl)-1-phenyl-9 H-pyrido[3,4-b]indole-3-carboxylate from the starting material L-tryptophan in good yield. The structure of the synthesised compound was established by 1H-NMR, 13C-NMR, and mass spectrometry, as well as single crystal X-ray diffraction analysis. The synthesised compound was screened for it's in vitro anticancer activity against four human carcinoma cell lines by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that the synthesised compound generated a low IC50 value of 14.2 µM in human chronic myelogenous leukaemia (CML) K562 cell line, suggesting that this compound has the potential to be developed further for its anticancer activity.

Overexpression of functional human FLT3 ligand in Pichia pastoris

Growth factors and cytokines including FLT3 Ligand (FLT3L) have many applications in cellular and molecular biology studies, and also as therapeutical agents. FLT3L was expressed in Pichia pastoris through cloning from human leukemic K562 cell line as well as using a codon-optimized synthetic construct. Codon adaptation index (CAI) was increased from 55 in the native sequence up to 95 after optimization. Significant changes occurred in the codons for Pro, Arg, Leu and Ser toward the favored codons in P. pastoris. Both forms of expressed FLT3L (from the native and optimized sequences) were capable of stimulating proliferation of the FDC-P1 cells expressing human wild-type FLT3 (FD–FLT3–WT). Sequence optimization resulted in 55-fold increase in the yield of active FLT3L (290 μg/mL of product in the crude supernatant). Amino acid residues 27–162 are sufficient for the biological function of human FLT3L. Pichia pastoris is a highly efficient and cost-effective system for expression of endotoxin-free FLT3L; however, codon optimization is necessary for its optimal expression.

A novel antitumor compound nobiliside D isolated from sea cucumber (Holothuria nobilis Selenka).

An anticancer compound, triterpene glycoside, was isolated from Holothuria nobilis Selenka. Its chemical structure and configuration were determined by two-dimensional nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The novel active compound was identified as nobiliside D, with the molecular formula C40H61O17SNa and chemical name 3-O-[-β-D-pyranosyl (1-2)-4'-O-sulfon-ate-β-D-xylopyranosyl]-alkoxy-9-ene-3β, 12α, 17α, 25β-4 alcohol. An antitumor test was performed using xCELLigence Real-Time Cell Analysis. Nobiliside D exhibited inhibitory effects on human leukemic cell line K562, human leukemia cell line U937, human lung cancer cell line A-549, human cervix carcinoma cell line HeLa, human breast cancer cell line MCF-7 and human liver carcinoma cell line HepG2. Nobiliside exhibited the greatest inhibitory effect on K562 and MCF-7 cells with an IC50 of 0.83±0.14 and 0.82±0.11 µg/ml, respectively. When human tumor cell lines K562 and MCF-7 were treated by nobiliside D (0.5 µg/ml) for 24 h, 45.8% of K562 cells and 58.7% of MCF-7 cells were apoptotic, whereas only 0.5% of un-treated control cells were apoptotic. These data indicate the compound should offer potential as a novel drug for the treatment of a range of cancers.

Ethaselen: a novel organoselenium anticancer agent targeting thioredoxin reductase 1 reverses cisplatin resistance in drug-resistant K562 cells by inducing apoptosis.

It has been reported that Ethaselen shows inhibitory effects on thioredoxin reductase (TrxR) activity and human tumor cell growth. In order to find an efficient way to reverse cisplatin resistance, we investigated the reversal effects of Ethaselen on cisplatin resistance in K562/cisplatin (CDDP) cells that were established by pulse-inducing human erythrocyte leukemic cell line K562, which are fivefold more resistant to cisplatin compared to K562 cells. The morphology and growth showed that the adhesion of K562/CDDP further decreased while the cell volume increased. The proliferation of K562/CDDP is strengthened. The antitumor activities in vitro were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and combination index (CI), showing the significant synergic effects of cisplatin and Ethaselen. Focusing on apoptosis, a series of comparisons was made between K562 and K562/CDDP. Cisplatin induced higher reactive oxygen species (ROS) generation in K562 and subsequently induced the formation of mitochondrial permeability transition pores (PTPs). In addition, cisplatin increased the ratio of Bax to Bcl-2 in K562, which can influence the mitochondrial membrane permeability. PTP formation and mitochondrial membrane permeabilization eventually resulted in the release of cytochrome c and activation of the Caspase pathway. However, these effects were not clearly seen in K562/CDDP, which may be the reason for the acquired CDDP resistance. However, Ethaselen can induce a high level of ROS in K562/CDDP by TrxR activity inhibition and increased ratio of Bax to Bcl-2 in K562/CDDP by nuclear factor κB (NF-κB) suppression, which subsequently induces the release of cytochrome c in K562/CDDP. This response is partly responsible for the reversal of the cisplatin resistance in K562/CDDP cells.

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.