Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state.

Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4ß1 and α5ß1 and show that the low-affinity states bind substantially faster than the high-affinity state. On- and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low-affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high-affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by 'inside-out signaling.'

Cytotoxic anthracycline and antibacterial tirandamycin analogues from a marine-derived Streptomyces sp. SCSIO 41399.

Aranciamycin K (1) and isotirandamycin B (2) were isolated from a marine-derived Streptomyces sp. SCSIO 41399, along with the previously reported four anthracycline derivatives (3-6), and two known tirandamycin derivatives (7 and 8). Their structures including absolute configurations were determined by extensive analysis of their spectroscopic analysis and ECD calculation method. Most of the isolated compounds were tested for their cytotoxic, antibacterial, and antifungal activities. Compounds 2, 7 and 8 displayed potent bacteriostatic effects against Streptococcus agalactiae with MIC values of 11.5, 5.9 and 5.7 µM, respectively. Besides, compounds 3, 5 and 6 exhibited moderate in vitro cytotoxic activities against the K562 cell lines with IC50 values of 22.0 ± 0.20, 1.80 ± 0.01 and 12.1 ± 0.07 µM, respectively.

Mechanical phenotyping of K562 cells by the Micropipette Aspiration Technique allows identifying mechanical changes induced by drugs.

Mechanical properties of living cells can be used as reliable markers of their state, such as the presence of a pathological state or their differentiation phase. The mechanical behavior of cells depends on the organization of their cytoskeletal network and the main contribution typically comes from the actomyosin contractile system, in both suspended and adherent cells. In the present study, we investigated the effect of a pharmaceutical formulation (OTC - Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the inflammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells. Here we show that by measuring the mechanical properties of cells exposed to OTC for different incubation times, it is possible to infer modifications induced by the formulation to the actomyosin contractile system. We emphasize that this system is involved in the first stages of the apoptotic process where an increase of the cortical tension leads to the formation of blebs. We discuss the possible relation between the observed mechanical behavior of cells aspirated inside a micropipette and apoptosis.

[Influence of HMGB1/MAPK/m-TOR signaling pathway on cell autophagy and chemotherapy resistance in K562 cells].

OBJECTIVE: To observe the effect of high-mobility group box 1 (HMGB1) on autophagy and chemotherapy resistance in human leukemiacell line (K562) cells, and to explore the underlying mechanisms.
 Methods: The K562 cells were cultured in vitro and divided into 6 groups: a chemotherapeutic group, a chemotherapeutic control group, a HMGB1 preconditioning group, a HMGB1 preconditioning control group, a HMGB1 siRNA group and a siRNA control group. The chemotherapeutic group was further divided into a vincristine (VCR) group, an etoposide (VP-16) group, a cytosine arabinoside (Ara-C) group, a adriamycin (ADM) group and a arsenic trioxide (As2O3) group. The cell activity was evaluated by cell counting kit-8. The protein levels of HMGB1, microtubule-associate protein1light chain3 (LC3), AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) were determined by Western blotting. The level of serum HMGB1 was evaluated by enzyme-linked immunosorbent assay (ELISA). The autophagy was examined by monodansylcadaverine staining and observed under transmission electron microscopy.
 Results: Compared with the control group, the cell activity was significantly decreased and the level of serum HMGB1 was significantly increased in the chemotherapeutic (VCR, VP-16, Ara-C, ADM and As2O3) groups (all P<0.05). Compared with the control group, the cell activity and the level of serum HMGB1 were significantly increased in the HMGB1 preconditioning group (both P<0.05). Compared with the siRNA control group, the cell activity and the level of serum HMGB1 were significantly decreased in the HMGB1 siRNA group (both P<0.05). Compared with the control group, the expression of LC3-II and the formation of autophagic bodies were increased in the HMGB1 preconditioning group (both P<0.05), the p-AMPK expression was increased and p-mTOR expression was decreased (both P<0.05).
 Conclusion: HMGB1 can increase the autophagy and promote chemotherapy resistance through the pathway of AMPK/m-TOR in K562 cells.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene.

Arecoline-induced death of human leukemia K562 cells is associated with surface up-modulation of TNFR2.

The goal of the present study is to explore the contribution of tumor necrosis factor-α (TNFα)-related pathway to the cytotoxicity of arecoline on human leukemia K562 cells. Arecoline treatment induced death of K562 cells and increased surface expression of TNFα, TNFR1, and TNFR2. Unlike that of TNFR1 mRNA, transcriptional levels of TNFα and TNFR2 mRNA increased in arecoline-treated cells. Moreover, arecoline-induced down-regulation of ADAM17 maturation was involved in surface up-modulation of TNFR1, TNFR2, and TNFα. Arecoline-elicited increase in intracellular Ca(2+) concentration was responsible for JNK/c-Jun pathway activation and ERK inactivation. Abolition of JNK/c-Jun pathway suppressed arecoline-induced increase in transcriptional level of TNFα and TNFR2 mRNA. TNFα and TNFR2 promoter luciferase activity and chromatin immunoprecipitating analyses revealed that c-Jun increasingly bound with TNFα and TNFR2 promoter upon arecoline treatment. Over-expression of constitutively active MEK1 abolished the effect of arecoline on suppressing ADAM17 maturation. Pretreatment with TNFR2 antibody abrogated arecoline-induced increased susceptibility of K562 cells for the cytotoxicity of TNFα and arecoline-induced cell death. Taken together, our data suggest that up-modulation of TNFR2 surface expression is associated with arecoline-induced death of K562 cells.

Differential gene expression profiles of human leukemia cell lines exposed to benzene and its metabolites.

Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. Benzene toxicity is likely mediated through metabolites induced by means of multiple pathways. Although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying leukemogenesis is not fully understood. The aim of this study was to analyze the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells that had been exposed to benzene and its metabolites. This was carried out using whole human genome oligonucleotide microarrays to ascertain potential biomarkers. Genes that were differentially expressed (>1.5-fold and p-values <0.05) after exposure to benzene (BZ), hydroquinone (HQ), and 1,4-benzoquinone (BQ) were then classified with GO, KEGG and GSEA pathway annotation. All genes that were identified were then functionally categorized as being involved in the cell cycle, the p53 signaling pathway, apoptosis, the MAPK signaling pathway, or the T cell receptor signaling pathway. Functionally important genes were further validated by means of real-time RT-PCR. The results showed that EGR1, PMAIP1, AR, CCL2, CD69, HSPA8, SLC7A11, HERPUD1, ELK1, and MKI57 genes altered their expression profiles. Similar expression profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, gene expression profiles along with GO, KEGG and GSEA pathway annotation analysis have provided an insight into the leukemogenesis as well as highlighted potential gene-based biomarkers of human leukemia cell lines when they are exposed to benzene and its metabolites.

Inhibitory mechanism of pure curcumin on Wilms' tumor 1 (WT1) gene expression through the PKCα signaling pathway in leukemic K562 cells.

The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells.

High glucose increases susceptibility to oxidative-stress-induced apoptosis and DNA damage in K-562 cells.

AIM: The study was carried out to evaluate the effect of several substrates on oxidative stress induced apoptosis and in K-562 cells. METHODS: Glucose at 5, 11 and 30 mM concentrations was tested, as well as 5 mM glutamine and 5 mM fructose. The cells were exposed to tert-butylhydroperoxide (tBH) and apoptotic cells were evaluated by flow cytometry with FITC-Annexin V and propidium iodide. The effect of glucose concentration on DNA damage was evaluated using hydrogen peroxide and electrophoretic "DNA comets" assay at 5 mM and 30 mM glucose concentrations. RESULTS: The exposure of cells to tBH resulted in increased number of apoptotic cells, and this effect was prevented by administration of an antioxidant - N-Acetyl cysteine. Rising concentrations of glucose added to the toxic effect of tBH; we also observed some toxic effect of fructose and no effect of glutamine. We found higher susceptibility to hydrogen peroxide induced DNA damage with 30 mM glucose concentration. CONCLUSION: Hyperglycemia increases the cell's susceptibility to oxidative stress and it also amplifies oxidative DNA damage. Glutamine - when used as a sole energetic substrate - showed no protective effect against oxidative stress.

Involvement of annexin A1 in multidrug resistance of K562/ADR cells identified by the proteomic study.

Multidrug resistance (MDR) to chemotherapy is a significant barrier to the effective treatment of chromic myeloid leukemia (CML). In an attempt to identify more factors associated with MDR for an understanding of the mechanism, we first established an adriamycin (ADR)-resistant human erythroleukemia cell line K562/ADR by stepwise selection in vitro using ADR. Besides the elevated resistance to ADR, the K562/ADR cells also showed significantly increased crossed-resistance to vincristin and Gleevec, compared to the parental K562 cells. Then we compared the global protein profiles between K562 and K562/ADR cells. Following two-dimensional gel electrophoresis and image analysis, some of the proteins with different levels between the two cell lines were identified by MALDI TOF/TOF mass spectrometry and Western blot analysis. The differentially expressed proteins were classified into groups based on their functions: calcium-binding proteins, chaperones, metabolic enzymes, proteins related to protein synthesis or DNA synthesis, and proteins related to signal transduction. In particular, ANXA1, a protein that was downregulated in K562/ADR, was analyzed further for its involvement in MDR by transfection and subsequent assays. The functional validation showed that the downregulated ANXA1 expression contributes considerably to the observed drug resistance in K562/ADR cells. These data will be valuable for further study of the mechanisms of MDR and may reveal a potential new diagnostic marker to chemotherapy.

Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.

Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0)39-globin mutation causing beta-thalassaemia.

[Expansion of human natural killer cells ex vivo].

AIM: To obtain sufficient numbers of pure and activated human primary NK cells for potential clinical application. METHODS: PBMC based ex vivo expansion of natural killer cells was set up. The expansion was initiated by co-culture of PBMC and stimulator cells (irradiate genetic modified K562 cells) in RPMI1640 medium with 1 000 U/mL IL-2. Genetic modified K562 cells 'K562D3' were prepared by expressing IL-15, 4-1BBL and IL-18 on K562 cell membrane. RESULTS: An average of 500 fold expansion of CD56(+)CD3(-) cells was observed after 3 weeks of co-culture. The NK cells population could reach 93% after expansion, comparing with 7% before expansion. The expanded NK cells lysed 95% of K562 targets in a 5:1 effector to target ratio. IL-15/4-1BBL bound K562 stimulatory cells could expand same fold NK cells as K562D3, but the cytotoxicity of NK cells expanded by 'K562D3' was 10% higher. CONCLUSION: The described method is a simple and efficient way for the expansion of human NK cells.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. II. Effect of anticancer drug doxorubicin].

The induction of apoptosis in K562 cells by doxorubicin (DR) was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed death. Here we have shown for the first time that proteasomes isolated from the nuclei of control and induced K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that trypsin- and chymotrypsin-like, and the endoribonuclease activities of nuclear 26S proteasomes are affected under influence of DR on K562 cells. Treatment of K562 cells with DR leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNase activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasomes population in undergoing apoptosis K562 cells which is manifested by changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. I. Effect of glutathione-depleting agent, diethylmaleate].

Here we have studied changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed cell death. Apoptosis in proerythroleukemic K562 cells was induced by glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and induces K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. We observed trypsin- and chymotrypsin-like activities on nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) to be changed under effect of DEM on K562 cells. Treatment of K562 cells with DEM leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNAse activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasome population in undergoing apoptosis K562 cells which is manifested by the changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

Iron deprivation blocks multilineage haematopoietic differentiation by inhibiting induction of p21(WAF1/CIP1).

Iron is required for the differentiation of HL-60 cells along the monocyte lineage in vitro, reflecting a requirement for iron in the transcriptional induction of the p21(WAF1/CIP1) gene. To determine if the same requirement holds true for differentiation in other cell lineages and for primary human CD34(+) bone marrow precursor cells, we induced granulocyte differentiation by treating HL-60 cells with dimethyl sulphoxide, and erythroid or megakaryocytic differentiation by treating K562 cells with butyrate or phorbol myristate acetate, respectively. Nitro blue tetrazolium reduction, expression of haem, or expression of CD41 was used to assess granulocytic, erythroid, or megakaryocytic differentiation respectively. Purified CD34(+) cells were cultured with granulocyte/macrophage-colony stimulating factor and stem cell factor to induce myelomonocytic differentiation. Iron deprivation was induced by desferrioxamine. p21(WAF1/CIP1) antisense oligonucleotides were used to inhibit p21 expression. Iron deprivation blocked p21 induction as judged by real-time polymerase chain reaction assays. In addition, both iron deprivation and p21 antisense blocked CD34(+) cell differentiation. These observations were not explained by induction of widespread apoptosis under conditions of iron deprivation. We concluded that both iron and functional p21(WAF1/CIP1) are required for in vitro differentiation of human haematopoietic precursors along multiple cell lineages.

[Specificity of changes in proteasome properties in diethylmaleate-induced apoptosis of K562 cells].

The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.

BaG, a new dimeric metalloproteinase/disintegrin from the Bothrops alternatus snake venom that interacts with alpha5beta1 integrin.

The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin-binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-beta-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin-ligand interactions.

DNA topoisomerase II inhibitor, etoposide, induces p21WAF1/CIP1 through down-regulation of c-Myc in K562 cells.

BACKGROUND: Anticancer agents modulate gene expression and these changes are essential for tumor cell killing. To investigate the mechanism by which etoposide acts as an anticancer agent, the relationship between p21WAF1/CIP1 (p21) and c-Myc was studied. MATERIALS AND METHODS: K562 cells with and without ectopic c-Myc expression were studied. Apoptosis was detected using propidium iodide and Hoechst 33342 double staining. The c-Myc and p21 levels were studied by RT-PCR and immunoblot. The p21 promoter (from -205 to +67) was investigated by the luciferase reporter gene assay. RESULTS: Ectopic c-Myc-expressing K562 (K562/c-Myc) cells showed more extensive apoptosis than K562 cells after continuous exposure to 200 microM etoposide for 24 hours. During this treatment, p21 expression was not observed in K562/c-Myc cells, and the expression of c-Myc and p21 was mutually exclusive. Etoposide activated the p21 promoter in a concentration-dependent manner, and etoposide-induced luciferase activity was suppressed by co-transfection of c-Myc. CONCLUSION: p21 promoter activity was repressed by c-Myc in proliferating K562 cells, and detoposide-induced down-regulation of c-Myc released this suppression, resulting in the induction of p21.

RB18A, whose gene is localized on chromosome 17q12-q21.1, regulates in vivo p53 transactivating activity.

Among the different cellular factors that regulated p53 functions, we previously identified (P. Drane et al., Oncogene, 15: 3013-3024, 1997) RB18A, a new gene whose encoded Mr 205,000 protein interacted in vitro, through its COOH-terminal domain, with p53. Therefore, we analyzed the in vivo role of RB18A by measuring its effect on the transactivating activity of p53 on physiological promoters. We herein demonstrated that RB18A, which interacted also in vivo with p53, activated Bax promoter and inhibited p21Waf1 or IGF-BP3 promoters. In addition, fluorescence in situ hybridization mapping led to localizing the RB18A gene on chromosome 17q12-q21.1, loci associated with human cancers. This is the first demonstration that in vivo RB18A, in a protein-protein interaction, regulates p53 transactivating activity.