Human Osteosarcoma Saos-2 Cells Research Articles

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.

Induction of contact-dependent endothelial apoptosis by osteosarcoma cells suggests a role for endothelial cell apoptosis in blood-borne metastasis.

Although tumour cells are believed to migrate between endothelial cells early in metastasis, the possibility remains that endothelial apoptosis may also contribute to the tumour's breach of the vascular barrier. Although seemingly inconsistent with tumour angiogenesis, one publication describes the induction of contact-dependent apoptosis in cultured endothelium by tumour cells. The cell culture data are, however, open to challenge on technical grounds while there are no confirmatory reports. The present paper describes experiments overcoming these limitations. SAOS-2 human osteosarcoma cells and two rat carcinoma cell lines were co-cultured with human umbilical vein endothelial cells (HUVECs) and cultures labelled by surface lectin histochemistry for endothelium. The HUVEC culture density was determined and SAOS-2 cells, but not rat carcinoma cells, were found significantly to reduce HUVEC survival despite the release of potent growth factors as determined in separate experiments with tumour cell conditioned medium. Lectin labelling combined with light microscopy, transmission electron microscopy, flow cytometry for both lectin binding and DNA content, and DNA gel electrophoresis of SAOS-2/HUVEC co-cultures revealed extensive HUVEC apoptosis. These findings indicate contact-dependent endothelial apoptosis by SAOS-2, while this activity appeared weaker and overwhelmed by HUVEC proliferation with rat carcinoma cells. Importantly, this study supports the suggestion that endothelial apoptosis may be important for metastasis and suggests a complex interplay between endothelial proliferation and apoptosis in tumours.

Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.

Lycopene II--effect on osteoblasts: the carotenoid lycopene stimulates cell proliferation and alkaline phosphatase activity of SaOS-2 cells.

We explored the possibility that lycopene, a carotenoid that is abundant in tomatoes, has effects on proliferation and differentiation of osteoblasts, the cells responsible for bone formation. Human osteoblast-like osteosarcoma SaOS-2 cells were cultured for 24 hours, after which varying doses of a water-dispersible microemulsion preparation of lycopene or vehicle of the same dilution were added. The cells were further cultured for 24 to 144 hours, and then the cell numbers were counted. Lycopene at 10(-6) and 10(-5) M had significant stimulatory effects on cell numbers, compared with the corresponding vehicle treatment, at all time points from 24 to 144 hours. The effects of lycopene on activity of the differentiation marker alkaline phosphatase activity in the absence or presence of dexamethasone were shown to be dependent on the stage of cell differentiation. This is the first report on the effects of lycopene on osteoblasts of human origin; the results may have important applications in the prevention of osteoporosis.

Targeted gene delivery to human osteosarcoma cells with magnetic cationic liposomes under a magnetic field

Gene delivery using cationic liposomes results in relatively low transfection, especially under in vivo conditions. This system, however, can overcome some of the problems associated with viral delivery systems. The present study was carried out in order to improve the transfection efficiency of cationic liposomes by preparing magnetic cationic liposomes (MCL). Small MCL approximately 40 nm in diameter and incorporating one or two magnetite particles were prepared with phosphatidylethanolamine and 3beta-[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol. The efficiency of MCL in gene delivery was evaluated by using plasmid DNA containing a luciferase reporter gene and human osteosarcoma Saos-2 cells. Without a magnetic field, maximum luciferase activity was observed when DNA was mixed with MCL at a 1:5 ratio and incubated with cells for 6 h. Under a magnetic field, maximum luciferase activity was achieved by 30-min magnetic induction. This improvement in transfection efficiency by magnetic induction was approximately 3.5-fold. The feasibility of this active transgenic system was further shown by measuring apoptosis rates after transfection of the p53 gene to Saos-2 cells. Apoptosis rates increased to 18.9% from 2.4% by magnetic induction. In conclusion, a gene delivery system including MCL and magnetic induction was found to achieve rapid and enhanced gene delivery in vitro. Such a gene delivery system may be applicable under in vivo conditions, and is expected to offer numerous clinical advantages.

Modulation of multidrug resistance-associated protein 1 (MRP1) by p53 mutant in Saos-2 cells

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancer and the mutation is correlated with a poor prognosis in cancer therapy. Upregulation of multidrug resistance-associated protein 1 (MRP1) and increase in drug resistance have been found to be induced by p53 mutation. Human osteosarcoma Saos-2 cells, a p53-null cell line, was transfected with p53 with mutations at codon 143 (V to A), 175 (R to H), 248 (R to W), 273 (R to H) and 281 (D to G). Among the different transfectants, overexpression (about 42-fold) of MRP1 was detected in p53-R175H cells. Furthermore, the p53-R175H cells were 2.5-fold more resistant to doxorubicin (DOX) and had a 4-fold greater DOX efflux rate than the control cells 1 h after DOX treatment. Transfection with antisense MRP1 oligonucleotides demonstrated a DOX sensitization effect (about 2-fold) in p53-R175H transfectants but not in control cells. In addition, transfection with antisense p53 oligonucleotides greatly suppressed MRP1 expression and reversed DOX resistance in p53-R175H cells but had no effect in control cells. The results suggested that p53-R175H might induce MRP1 expression and DOX resistance in cells.

Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase

The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase-positive cells and alternative lengthening of telomeres (ALT) in telomerase-negative cells. We report here that telomere maintenance in the telomerase-positive human ovarian SKOV-3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase-positive cell line (i.e. human pharynx FaDu cells) and the telomerase-negative human osteosarcoma Saos-2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization-based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3′-azido-deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV-3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of ∼80%) and by expression of antisense hTR (complete inhibition in SKOV-3 cells and maximal inhibition of ∼80% in FaDu cells). However, treatment with telomerase inhibitors resulted in ∼50% telomere shortening in FaDu cells but had no effect on SKOV-3 nor Saos-2 cells. SKOV-3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos-2 cells. Collectively, these results suggest the existence of a telomerase-independent mechanism of telomere maintenance in the telomerase-positive SKOV-3 cells.

Identification and Characterization of Soluble Isoform of Fibroblast Growth Factor Receptor 3 in Human SaOS-2 Osteosarcoma Cells

We have previously reported the alternatively spliced transcripts of fibroblast growth factor 3 (FGFR3 ATs and MTs) derived by aberrant splicing and usage of cryptic splicing sites. Here, we describe a soluble variant of FGFR3 (FGFR3 AT-III) arising from skipping exons 8, 9, and 10 in human SaOS-2 osteosarcoma cell. This splicing event leads to the generation of an mRNA encoding a FGFR3 in which the COOH-terminal portion of the Ig-like-III domain and transmembrane domain are deleted while the remainder of the mature molecule is fused in-frame to the COOH-terminal cytoplasmic kinases domains. Sf9 cells transfected with the corresponding cDNA express the soluble form of FGFR3 AT-III into the condition medium and its secreted form was able to bind both FGF-1 and FGF-2 leading to loss of ligand binding specificity. These results indicate that the FGFR3 AT-III mRNAs are transcribed due to exon skipping with altered ligand binding specificity. These results suggest that the presence of soluble transcripts of FGFRs gene is a common feature due to mRNA splicing and this splicing plays an important role in the regulation of FGFRs function.

Differential expression of Notch genes in human osteoblastic cells

Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types. In the present study we investigated the expression of four Notch genes in primary human osteoblasts and in human osteoblastic osteosarcoma cell line SaOS-2 by RT-PCR. We found a strong constitutive expression of Notch-1 and a weak constitutive expression of Notch-2 in both cell types. After stimulation with Dexamethasone or Vitamin D(3), two factors known to induce differentiation in osteogenic cells, both Notch receptors were downregulated, however, with a different time course. Notch-1 and Notch-2 showed a transient induction after 2 days and a decrease after 7 days in osteoblasts and after 28 days in SaOS-2 cells. Notch-4 expression could only be detected after stimulation with Dexamethasone and Vitamin D(3). However, in osteoblasts a transient induction after 2 days could be detected in osteoblasts, whereas Notch-4 expression increased after 14 and 28 days in SaOS-2 cells. In contrast, Notch-3 was not expressed in human osteoblasts and SaOS-2 osteosarcoma cells. These data show, that Notch genes are expressed in human osteoblastic cells and that the expression is differentially regulated upon stimulation with osteogenic factors.

ヒト骨芽細胞のオカダ酸誘導アポトーシスと核内蛋白の変化

Alterations of nucleolar proteins during apoptosis are not well understood. We examined the AgNORs in apoptotic cells using double staining with Hoechst 33342 and silver nitrate. Apoptosis was induced by treatment of the human osteosarcoma cell line, Saos-2 cells and MG63 cells with okadaic acid. AgNORs, visible as dots in the nucleus of the control cells, disappeared from the apoptotic nuclei in both cell lines. Proteins prepared form Saos-2 cells treated with 20 nM okadaic acid for various time periods were prepared and subjected to SDS-PAGE followed by transferring to transfer membranes and stained with silver nitrate. Two major bands, 110 kDa and 37 kDa, were detected in the proteins obtained from the control cells. The 110 kDa protein level was decreased in the apoptotic cells. However, an additional band, 80 kDa, appeared and the level of the 80 kDa protein increased in the proteins prepared from okadaic acid-induced apoptotic cells. The reaction intensity of the 37 kDa protein did not change in the apoptotic cells. Cellular fractionation of Saos-2 cells was done in the control and apoptotic cells. Two major bands, 110 kDa and 37 kDa, were detected in the nuclear fraction prepared from the control cells. The 80 kDa protein was detected and the 110 kDa protein level was decreased in the nuclear fraction from the apoptotic cells. In the cell-free apoptotic system, the 80 kDa protein also was detected while the 110 kDa protein was decreased in the proteins extracted from the nuclei of Saos-2 cells, which underwent apoptosis in vitro. The changes of these AgNOR proteins in vitro were identical in the nuclei prepared from MG63 cells. We deduced that the 110 kDa and 37 kDa proteins are nucleolin and nucleophosmin, respectively, from their AgNORs staining features including their molecular weights. The 80 kDa protein may be the cleavage product of the 110 kDa protein. Our results indicate that the nucleolar AgNOR proteins are associated with the induction of DNA fragmentation or the final active phase of apoptosis. In the present study, We also examined the cytolocalization of AgNORs, nucleolin, and nucleophosmin and the expression of these proteins in Saos-2 cells by immunofluorescent technique, Western blot analysis and RT-PCR method. Staining pattern of nucleolin and nucleophosmin in Saos-2 cells and MG63 cells is similar to that of the AgNORs. Nucleolin and nucleophosmin were specifically stained as dots in the nucleus in both cell lines. The dual fluorescence image revealed that nucleolin and nucleophosmin represent the same localization in nucleolus. In the apoptotic Saos-2 cells nucleolin and nucleophosmin redistributed to the whole nucleus. The levels of nucleolin decreased in the apoptotic cells whereas that of nucleophosmin did not change. The levels of mRNAs of nucleolin and nucleophosmin did not change in the apoptotic cells. These findings suggest that degradation of nucleolin is regulated at the post-translational level.

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.

PRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase

This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.

Modulation of osteosarcoma cell growth and differentiation by silane‐modified surfaces

The effects of growing the Saos-2 human osteosarcoma cell line onto surfaces containing -CH3, -OH, -COOH, -NH2, and C6H5 groups obtained by silane modification were examined. These cells were used because of the great importance of bone cells in many aspects of biomaterials research. Silane-modified surfaces were characterized by contact angle measurements and, subsequently, surface energies were calculated. Cells grown on clean glass, as well as those grown on glass surfaces containing the functional groups cited above, were examined by light and scanning electron microscopy and assessed for their growth characteristics (i.e., determination of cell number and Ki67 antigen expression). The data presented seemed to indicate that if Saos-2 cells are grown on silane-modifed surfaces containing the methyl (CH3), hydroxyl (OH), and phenyl (C6H5) functional groups, their proliferation is slowed down while growth of these cells on glass surfaces modified with amino (NH2) and carboxyl (COOH) groups did not significantly affect growth. Once it was demonstrated that these three functional groups induce significant variations in proliferation, cells grown on these surfaces were also tested for apoptosis and expression of important markers of bone cell differentiation (i.e., osteonectin and osteopontin) by flow cytometry and eventual rearrangement of these markers by fluorescence microscopy. The data suggested that growth of Saos-2 cells on CH3 induces the most evident morphological changes while growth of these cells on OH and C6H5 brings about the greater variations in osteonectin and osteopontin. We hypothesized that these changes are indicative of an increase in differentiation of Saos-2 cells when grown on the OH and C6H5 groups. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 338–349, 2001

A bone replaceable artificial bone substitute: osteoinduction by combining with bone inducing agent.

Bone inducing agent (BIA) isolated from Saos-2 human osteosarcoma cells was added to an artificial bone substitute composed of 980 degrees C-heated carbonate apatite (CAp) and Type I atelocollagen (AtCol) extracted from bovine tail skins (88/12 in wt/wt %), and a CAp-AtCol-BIA substitute was prepared as an osteoinductive bone substitute. Rat calvaria osteoblasts treated by the isolated BIA demonstrated significantly increased alkaline phosphatase (ALP) activity after 3 days (p < 0.05). In vitro cell attachment and proliferation and ALP activity were investigated for the bone substitute combined with BIA. Osteoblasts cultured onto the surface of the CAp-AtCol-BIA substitute demonstrated remarkable morphological changes such as radial spreading, flattening, and projecting filopodia after 5 days. In comparison with the substitute without BIA, osteoblasts grown in the BIA-combined CAp-AtCol substitute expressed significantly increased proliferation and ALP activity, respectively (p < 0.05). Both the substitutes combined with and without BIA were implanted into artificial defects created in rabbit radii. After 4 weeks, the CAp-AtCol-BIA substitute implanted lesion was completely replaced by regenerated host bone in radiological observation whereas the substitute without BIA was partially resorbed. No histologic abnormalities appeared in the substitute either with or without BIA.

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A 9C 9, 16D 9A 4 and 20A 12C 7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A 9C 9, and between 282 and 372 for 16D 9A 4 and 20A 12C 7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation.

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.

Modulation of osteosarcoma cell growth and differentiation by silane-modified surfaces.

The effects of growing the Saos-2 human osteosarcoma cell line onto surfaces containing -CH(3), -OH, -COOH, -NH(2), and C6H5 groups obtained by silane modification were examined. These cells were used because of the great importance of bone cells in many aspects of biomaterials research. Silane-modified surfaces were characterized by contact angle measurements and, subsequently, surface energies were calculated. Cells grown on clean glass, as well as those grown on glass surfaces containing the functional groups cited above, were examined by light and scanning electron microscopy and assessed for their growth characteristics (i.e., determination of cell number and Ki67 antigen expression). The data presented seemed to indicate that if Saos-2 cells are grown on silane-modifed surfaces containing the methyl (CH(3)), hydroxyl (OH), and phenyl (C6H5) functional groups, their proliferation is slowed down while growth of these cells on glass surfaces modified with amino (NH(2)) and carboxyl (COOH) groups did not significantly affect growth. Once it was demonstrated that these three functional groups induce significant variations in proliferation, cells grown on these surfaces were also tested for apoptosis and expression of important markers of bone cell differentiation (i.e., osteonectin and osteopontin) by flow cytometry and eventual rearrangement of these markers by fluorescence microscopy. The data suggested that growth of Saos-2 cells on CH(3) induces the most evident morphological changes while growth of these cells on OH and C6H5 brings about the greater variations in osteonectin and osteopontin. We hypothesized that these changes are indicative of an increase in differentiation of Saos-2 cells when grown on the OH and C6H5 groups.

The N-terminal domain of p73 interacts with the CH1 domain of p300/CREB binding protein and mediates transcriptional activation and apoptosis.

The newly identified p53 homolog p73 mimics the transcriptional function of p53. We have investigated the regulation of p73's transcriptional activity by p300/CREB binding protein (CBP). p73-p300 complexes were identified in HeLa cell extracts by cofractionation and coimmunoprecipitation assays. The p73-p300 interaction was confirmed in vitro by glutathione S-transferase-protein association assays and in vivo by coimmunoprecipitating the overexpressed p300 and p73 in human p53-free small-cell lung carcinoma H1299 or osteosarcoma Saos-2 cells. The N terminus but not the N-terminal truncation of p73 bound to the CH1 domain (amino acids [aa] 350 to 450) of p300/CBP. Accordingly, this p73 N-terminal deletion was unable to activate transcription or to induce apoptosis. Overexpression of either p300 or CBP stimulated transcription mediated by p73 but not its N-terminally deleted mutant in vivo. The N-terminal fragment from aa 19 to 597, but not the truncated fragment from aa 242 to 1700 of p300, reduced p73-mediated transcription markedly. p73-dependent transcription or apoptosis was partially impaired in either p300- or CBP-deficient human breast carcinoma MCF-7 or H1299 cells, suggesting that both coactivators mediate transcription by p73 in cells. These results demonstrate that the N terminus of p73 directly interacts with the N-terminal CH1 domain of p300/CBP to activate transcription.

P73 competes with p53 and attenuates its response in a human ovarian cancer cell line.

The transcriptional activity of the p53 tumor suppressor protein is crucial for the regulation of cell growth, apoptosis and tumor progression. The first identified p53 relative, p73, was reported to be monoallelically expressed in normal tissues. In some tumors, loss of heterozygosity was associated with overexpression of the silent allele. Human p73alpha was transfected into the wild-type p53-expressing human ovarian carcinoma cell line A2780. Unlike human osteosarcoma Saos-2 cells, A2780 cells could tolerate hyperexpression of p73alpha and clones over-expressing p73alpha could be isolated. No p53-p73 protein-protein interaction was found in these clones in co-immunoprecipitation experiments. Endogenous p53 transcriptional activity was markedly decreased both when p73 was integrated into the genome and in transient transfections using a reporter plasmid containing the p53 binding site linked to luciferase. Transient transfection of p73 with a mutation in the DNA-binding domain did not show these effects. The competition for p53 DNA binding by p73alpha was also evident in gel shift experiments. The results suggest that p73 can modulate p53 function by inhibiting its DNA binding and that overexpression of p73 in tumors might be a novel mechanism of inactivation of p53.

Aromatic Hydrocarbon Receptor Interaction with the Retinoblastoma Protein Potentiates Repression of E2F-dependent Transcription and Cell Cycle Arrest

Polyhalogenated aromatic hydrocarbons, of which 2,3,7, 8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR.RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.