Transcriptional control of p53 expression participates in the generation of appropriate levels of active p53 in response to mitogenic stimulation. This prompted us to study the role of a putative AP-1 and a NF-kB motif in the human p53 promoter for transcriptional regulation. We show that mutation of the AP-1 or the NF-kB motif abolishes transcription from the human p53 promoter in HeLa, HepG2 and adenovirus type 5 E1-transformed 293 cells. In comparison, mutation of the previously characterized Myc/Max/USF binding site in the human p53 promoter reduces the transcription rate ®vefold. The AP-1 motif in the human p53 promoter binds c-Fos and c-Jun and the NF-kB motif binds p50 NF-kB1 and p65 RelA . The cooperative nature of transcriptional activation by these factors was documented by repression of c-fos or NF-kB1 translation: Pretreatment of the cells with a cfos or p50 NF-kB1 antisense oligonucleotide suppresses transcription from the human p53 promoter completely. In addition, we show that (a) the level of endogenous p53 mRNA and (b) transcription from the strictly p53-dependent human mdm2 promoter are reduced in the presence of c-fos, c-jun, p50 NF-kB1 , p65 RelA or c-myc antisense oligonucleotides, underscoring the importance of these transcription factors for the expression of functional p53.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.

Bone morphogenetic proteins (BMPs) play a decisive role in bone development and osteogenesis. In the past they have been the subject of widespread research and clinical trials as stimulants of bone growth. Although recently recombinant human BMP-2 (rhBMP-2) has been chemically immobilized on implant surfaces leading to enhanced bone growth and accelerated integration in vivo, the non-covalent immobilization of proteins on metal surfaces is still poorly understood, since the oxide layers on metals like titanium, stainless steel or cobalt chromium alloys are poor adsorbents of proteins. Protein binding surfaces could either be generated by linking ionic groups (ion-exchange surface) or by coupling hydrophobic residues (hydrophobic interacting surface, HIS) to the surface. In this paper the preparation of protein adsorbing surfaces on titanium and cobalt chromium molybdenum alloy for the adsorption of rhBMP-2 and ubiquitin will be described. rhBMP-2 and ubiquitin are bound extremely tight to surfaces containing propyl or hexyl groups of a certain surface concentration and are slowly released over a range of at least 24 ± 100 days making such surfaces applicable as long-term drug delivery devices for enhancing bone growth or implant integration.Knochenmorphogenetische Proteine (BMPs) spielen eine entscheidende Rolle in der Entwicklung der Organismen und der Osteogenese. In der Vergangenheit waren sie in der Stimulierung des Knochenwachstums Thema zahlreicher Untersuchungen und klinischer Studien. Obwohl ku Èrzlich rekombinantes humanes BMP-2 (rhBMP-2), das auf Implantatoberfla Èchen chemisch immobilisiert wurde zu versta Èrktem Knochenwachstum und zur beschleunigten Integration in vivo fu Èhrten, ist die nicht-kovalante Immobilisierung von Proteinen auf metallischen Oberfla Èchen bisher nur wenig verstanden, da die Oxidschichten auf Metallen wie Titan, rostfreiem Stahl oder Kobalt-Chrom-Legierungen Proteine nur schlecht adsorbieren. Proteinbindende Oberfla Èchen ko Ènnen entweder durch die Kopplung von geladenen Moleku Èlen (Ionenaustauscheroberfla Èche) oder von hydrophoben Resten (hydrophob interagierende Oberfla Èchen, HIS) an die Oberfla Èche hergestellt werden. In dieser Arbeit wird die Herstellung von Protein adsorbierenden Oberfla Èchen auf Titan und einer Kobalt-Chrom-Molybda Èn Legierung fu Èr die Adsorption von rhBMP-2 und Ubiquitin beschrieben. RhBMP-2 und Ubiquitin werden auûerordentlich stark an Oberfla Èchen gebunden, die Propyl-oder Hexylgruppen bestimmter Oberfla Èchenkonzentration tragen und werden langsam u Èber einen Zeitraum von 24 ± 100 Tagen von den Oberfla Èchen wieder freigesetzt, so daû sich diese als langzeit ¹drug delivery devicesª zur Anregung des Knochenwachstums und zur Beschleunigung der Implantatintegration eignen.

The binding of rhBMP‐2 to its receptors, the signal transduction cascade and the final responses of bone cells, osteoprogenitor cells and derived cell lines is of high fundamental and clinical interest. In this report concentration‐response curves of the osteoblast cell line MC3T3‐E1 under influence of rhBMP‐2 was investigated. The biological response of the cells (corresponding to a down‐stream effect of the receptor state‐function) was monitored in pilot experiments by the MC3T3‐cell alkaline phosphatase‐induction test (MC3T3‐cell ALP‐induction test). It is shown that the MC3T3‐cell ALP‐induction test is a good tool for measuring biologically active recombinant human BMP‐2 (rhBMP‐2) in crude extracts of E. coli as well as in highly purified form. In addition this test is very sensitive to chemically induced structural changes of rhBMP‐2 such as those resulting from a radiolabeling of rhBMP‐2 by the Bolton‐Hunter procedure. The latter procedure reduces the biological activity of rhBMP‐2 by a factor of 3–4. The measured concentration‐response curves could all be non‐linearly fitted to a rectangular hyperbola. The half‐maximal saturation, K0.5, is found between 30–100 nM rhBMP‐2 (= 0.8–2.5 μg/ml). The effect of rhBMP‐2 shows a plateau i.e. maximal response at ca. 300–1000 nM rhBMP‐2 (= 8–25 μg/ml). The data thus indicate a non‐cooperative binding‐response behavior. This was unexpected since BMP‐2 binds simultaneously to two cooperating receptors of type 1 and type 2. However in the very low concentration range of employed rhBMP‐2 a variable response of the cells was measured so that a full exclusion of cooperativity cannot be concluded at the present time. This will be clarified by future experiments.

Previously it has been shown that recombinant human bone morphogenetic protein (rhBMP‐2) can be chemically immobilized by “anchor molecules” on titanium surfaces for serving as a drug delivery device. This opened the question of whether the insoluble immobilized rhBMP‐2 retained its activity in comparison to the same amount of soluble rhBMP‐2 included with the implant samples. Electropolished titanium miniplates (10 × 6 × 0.8 mm) were “surface‐enhanced” by a novel treatment with chromosulfuric acid and then coated with a total amount of 150–200 ng rhBMP‐2 prepared by recombinant technology. Periosteal flaps (7 × 20 mm) were detached and isolated from the anterior surface of the tibiae of adult rabbits and wrapped around the titanium sample plates which were then implanted in the M. gastrocnemius. In the first experimental group various controls without rhBMP‐2 were combined (n = 12). In the second experimental group implants with chemically immobilized rhBMP‐2 (n = 8) were compared with implants to which non‐immobilized soluble rhBMP‐2 was added (n = 8). Animals were sacrificed after 28 days and a quantitative evaluation was carried out by means of serial sections. Untreated control plates showed bone formation in 2/12 implants, rhBMP‐2 coated implants in 6/8 and implants with free rhBMP‐2 administered subperiostally in 8/8 cases. In the case of rhBMP‐2 coated implants the induced bone had direct contact to the implant in all cases while in the group with free administered rhBMP‐2 the bone had no contact to the implant in two cases, but was separated by a fibrous capsule. Bone volume, bone surface area, and trabecular number displayed no difference between the two rhBMP‐2‐groups. However, in the biocoated group a tendency to an increase in the bone‐implant contact area was evident. No differences in osteoid area, osteoid perimeter and eroded perimeter were detected. We conclude that in the case of non‐immobilized rhBMP‐2 there is the danger for formation of fibrous tissue between the implant and the newly formed bone and in addition the generation of ectopic bone at inappropriate places. In contrast chemically immobilized rhBMP‐2 does not have these drawbacks and at the same time displays a biological activity on surfaces similar to that of soluble rhBMP‐2 demonstrating that biomaterial surfaces can be tailored for a selective and specific interaction with the target tissue.

The osseointegration of long‐term implants is often incomplete such that gaps remain between the implant surface and the surrounding hard tissue. This study examines the effect of soluble recombinant human bone morphogenic protein 2 (rhBMP‐2) on gap healing and osseous integration. The effect of a single, intraoperative application of soluble rhBMP‐2 on the formation of new bone around titanium implants was studied. A total of 8 titanium‐alloy cylinders (Ti‐6Al‐4V) with a plasma spray coating (TPS; 400 μm thickness) were implanted into femoral condyles of mature sheep: rhBMP‐2 solution (1 μg) was pipetted into the 1 mm wide cleft around 4 implants; 4 further implants served as rhBMP‐2‐free controls. Two of these controls exhibited an additional calciumphosphate‐coating. The cleft around the implants served as testing zone to study the formation of new bone by microradiographical and histological analyses. The follow‐up periods were 4 and 9 weeks, respectively. A significant amount of new bone contacting the implants' surface was detected where rhBMP‐2‐solution had been used: In 50% a circumferential osseoinduction occurred within 4 weeks and a nearly complete osseointegration was observed after 9 weeks. In all cases bone formation was exaggerated and filled the spongiosa with compact bone. Time matched TPS‐controls and controls with calciumphosphate coating showed no notable formation of new bone. The results suggest that a single administration of soluble rhBMP‐2 into a bone cavity can augment bone formation and also osseointegration of titanium implants. Further investigations based on these findings are necessary to develop long‐term implants (e. g. joint replacements) with rhBMP‐2‐biocoating for humans.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) has been widely employed for the induction of bone growth in animal models and in clinical trials. Since rhBMP-2 is commercially available only to a very limited extent, we have prepared our own recombinant material for the current testing and for future applications. As yet, no toxicological data on rhBMP-2 has been reported to the knowledge of the authors. Therefore, it was considered necessary to apply the in vitro-micronucleus assay to our preparation to monitor a possible genotoxic effect. A total of approximately 24500 nuclei from V79 Chinese hamster cells and more than 40000 nuclei from human amniotic fluid fibroblast-like (AFFL) cells were screened for the frequency of micronuclei following incubation with 3-300 nM rhBMP-2, which covers the concentration range from initial biological activity to the begin of a maximal response in MC3T3-E1 cells. It can be concluded from the results that our preparations of recombinant human BMP-2 prepared in E. coli do not cause DNA damage in the concentration range tested.Key words: BMP-2, mutagenesis, toxicology, micronucleus, mitosis, cisplatin, V79 hamster Rekombinantes humanes Bone Morphogenetic Protein (rhBMP-2) ist bereits vielfach zur Induktion des Knochenwachstums in Tierexperimenten und klinischen Untersuchungen eingesetzt worden. Da jedoch rhBMP-2 auf dem Markt nur sehr beschränkt zu erhalten ist, stellen wir unser rhBMP-2 zu Testzwecken und für künftige Anwendungen selbst her. Interessanterweise ist den Autoren bisher kein Toxizitätstest über rhBMP-2 bekannt geworden. Daher schien es notwendig unsere Präparationen einem in vitro Mikrokerntest auf das Vorliegen einer möglichen Genotoxizität hin zu prüfen. Im Rahmen dieser Arbeit wurden etwa 24500 Kerne von chinesischen Hamsterzellen (V79) und mehr als 40000 Zellkerne von humanen "amniotic fluid fibroblast-like" (AFFL) Zellen ausgezählt und die Zellen nach dem Vorliegen von Mikrokernen untersucht. Dabei wurden die Zellen in Konzentrationen von 3-300 nM des rhBMP-2 inkubiert, welches einen Konzentrationsbereich vom Beginn einer biologischen Reaktion bis zur Maximalen Reaktionsweise der Osteoblastenzellinie MC3T3-E1 umfaßt. Aus den gewonnenen Ergebnissen schließen wir, daß unsere Präparationen von rhBMP-2 aus E. coli im genannten Konzentrationsbereich keine DNA-Schädigungen hervorrufen.

Transformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the unique spacer region of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of the spacer binds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a second spacer mutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1 diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awt or, least efficient, E1ASpm2. These properties of the spacer mutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1 (plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2 or Ad12 E1AdelCR2 (lacking the entire CR2) died within 6-10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes approximately 120 h. Moreover, in the established cell lines, the affinity of E1ASpm1 to p105-Rb was higher than with that of E1Awt. Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-unique spacer, whereas the hydrophobic stretch of six alanines in the spacer is required for stable transformation.