text:
Zinc Finger Transcription Factors Mediate High Constitutive Platelet-derived Growth Factor-B Expression in Smooth Muscle Cells Derived from Aortae of Newborn Rats*

Platelet-derived growth factor (PDGF) B-chain gene is differentially expressed in smooth muscle cells (SMCs) derived from the rat aortic wall. SMCs cultured from two week-old rats (pups) express high levels of PDGF-B mRNA, whereas cells isolated from three month-old rats (adults) express low levels of PDGF-B. Nuclear run-off experiments indicate that increased PDGF-B gene expression in pups is mediated, at least in part, at the transcriptional level. We used electrophoretic mobility shift assays and Western blot analysis to demonstrate that levels of Sp1 and Sp3, two zinc finger transcription factors which mediate basal expression of the PDGF-B gene, are elevated in pup nuclei compared with adult nuclei. The immediate-early transcription factor, Egr-1, which footprints the PDGF-B promoter, is also constitutively expressed in these cells. Transient transfection and binding studies show that these factors interact with a region in the proximal PDGF-B promoter key for basal expression in pup cells. Mutation of this proximal element in transfected pup cells attenuates reporter gene expression to levels observed in adult cells. Conversely, overexpression of Sp1 in adult cells augments PDGF-B promoter-dependent expression. Elevated PDGF-B expression in cultured newborn rat SMCs may therefore require high constitutive expression of a number of zinc finger transcription factors and their specific interactions with the proximal PDGF-B promoter.

in culture. Seifert et al. (3) observed over a decade ago that PDGF may be developmentally regulated in these cells. SMCs cultured from the aortae of 2-week-old rats (pups) (WKY12-22 cells) secreted almost 200-fold greater levels of PDGF than cells isolated from 3-month-old rats (adults) (WKY3M-22 cells). Subsequent Northern blot analysis determined that PDGF-B gene expression is at least 60-fold greater in cultured pup SMCs than adult cells (4). Evidence that differential PDGF-B expression is mediated at the level of transcription was obtained from nuclear run-off analysis (5). Several other genes, such as tropoelastin and ␣ 1 -procollagen (type I), were found to be overexpressed in pup cells relative to adult cells by cDNA library screening strategies. Cloning pup cells by dilution plating revealed that subpopulations expressed high levels of elastin, CYPIA1 and osteopontin mRNA (6). Cultured pup and adult SMCs also display marked differences in morphology. The epithelioid and cobblestone shape of pup cells contrasts with the spindle-like, elongated bipolar nature of adult SMCs (4).
Basal and inducible expression of the PDGF-B gene is mediated by nucleotide elements located approximately 40 bp upstream of the TATA box. We and others showed that Sp1 binds to the 5Ј-CCACCC-3Ј motif and drives basal expression in vascular endothelial (7) and osteosarcoma cells (8). This is consistent with in vivo footprinting studies which indicate that this region is occupied by nuclear factors in intact cells (9). The related zinc finger transcription factor, Sp3, also interacts with this region of the promoter and activates transcription (8,10). Egr-1, also known as TIS8, zif268, and NGFI-A (11), plays a positive regulatory role in PDGF-B promoter-dependent expression in cells exposed to phorbol esters or subjected to mechanical injury (12,13). Egr-1 binds to a cryptic recognition element in the PDGF-B promoter and can displace Sp1 from overlapping binding sites (13). This region also mediates inducible PDGF-B promoter-dependent gene expression in endothelial cells exposed to thrombin (14); however, functional transacting factors mediating this process have not yet been identified.
We hypothesized that the restricted expression of PDGF-B in subpopulations of cultured SMCs may be regulated by functional interactions of key zinc finger transcription factors with nucleotide elements in the proximal PDGF-B promoter. Here, we report that Sp1, Sp3, and Egr-1 are constitutively elevated in pup SMCs compared with adult cells. Promoter deletion and transient transfection analysis in pup cells revealed that the region bound by these factors is critical for basal expression directed by the PDGF-B promoter. Mutation of this region attenuates expression to levels comparable with those in adult cells. These findings provide mechanistic insights into the differential expression of PDGF-B gene in SMCs.

EXPERIMENTAL PROCEDURES
Cell Culture-Pup and adult SMC lines were cultured in Waymouth's MB752/1 medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum, 30 g/ml L-glutamine, 10 units/ml penicillin, and 10 g/ml streptomycin at 37°C and 5% CO 2 . Cultures were passaged every 3-4 days in 75 cm 2 flasks. Cells seeded for preparation of nuclear extracts were grown to 80% confluence and washed twice in phosphatebuffered saline (PBS), pH 7.4, then returned to the incubator in 1% growth medium for 24 h prior to extraction.
Transient Cell Transfections-SMCs were seeded in 100-mm tissue culture plates 2 days prior to transfection. When approximately 60% confluent, the cells were transfected with 10 g of the indicated promoter-reporter plasmid along with 2 g of pTKGH to correct for transfection efficiency. In overexpression studies, SMCs were cotransfected with 3 g of either Sp1-pcDNA3 (CMV promoter based), 5 g of Egr-1-pcDNA3, or pcDNA3 alone. Transfections were performed using Superfect (Qiagen). A precipitate was formed using 5 l of Superfect reagent/g of plasmid DNA and the transfection mix made up to 300 l with serum-and antibiotic-free Waymouth's medium. After incubation at 22°C for 10 min, 3 ml of complete Waymouth's growth medium was added, and the entire mixture was then added to cells previously washed once in PBS, pH 7.4. Following a 3-h incubation at 37°C in an atmosphere of 5% CO 2 , the precipitate was gently aspirated, and the monolayers were washed once with 4 ml of PBS, pH 7.4, prior to the addition of 10 ml of complete Waymouth's growth medium. For overexpression experiments, SMCs were refed with 2% Waymouth's medium. Two days post-transfection, the conditioned medium was collected for determination of soluble growth hormone by enzyme-linked immunosorbent assay (Bioclone Australia), and cell lysates were prepared for assessment of chloramphenicol acetyltransferase activity as described (7).
Preparation of Nuclear Extracts-SMCs were washed twice with PBS, pH 7.4, at 4°C and removed from the culture dish by scraping. The cells were pelleted by centrifugation at 1200 rpm for 10 min at 4°C. The pellet was resuspended in cold PBS, pH 7.4, and the suspension was transferred to Eppendorf tubes. Cells were repelleted in a microcentrifuge by spinning at 14,000 rpm for 20 s at 4°C. The cells were lysed by resuspending the pellet in an ice-cold Buffer A, which consisted of 10 mM HEPES, pH 8, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol (DTT), 200 mM sucrose, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, and 1 mg/ml aprotinin. After incubation on ice for 5 min, the suspension was recentrifuged and the nuclei were lysed in ice-cold Buffer C, which consisted of 20 mM HEPES, pH 8, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml aprotinin. Cellular debris was removed by centrifugation, and the supernatant containing DNA-binding proteins was combined with an equal volume of ice-cold Buffer D (20 mM HEPES, pH 8, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml aprotinin). Extracts were immediately frozen on dry ice and then stored at Ϫ80°C until use.
Electrophoretic Mobility Shift Assay (EMSA)-Binding reactions for gel shift assays were performed in 20 l of 10 mM Tris-HCl, 50 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM PMSF, 1 g of salmon sperm DNA (Sigma), 5% sucrose, 1 mg of poly(dI⅐dC)-poly(dI⅐dC) (Sigma), 32 P-labeled oligonucleotide probe (150,000 cpm), and 3 g of nuclear extract (determined by Bio-Rad Protein Assay). The reaction was incubated for 35 min at 22°C. In supershift studies, 2 l of the appropriate affinity-purified antipeptide polyclonal antibody (Santa Cruz Biotechnology) was incubated with the binding mix for 10 min before addition of the probe. These antibodies do not cross-react with the others' protein target. Bound complexes were separated from free probe by loading samples onto a 6% non-denaturing polyacrylamide gel and electrophoresing at 200 V for 2.5 h. The gels were vacuum dried at 80°C and subjected to autoradiography overnight at Ϫ80°C.

Certain Zinc Finger Transcription Factors
Are More Abundant in Cultured Pup Aortic SMCs than Adult SMCs-Previous investigations by our group (7, 13) and others (8,14,15) determined that PDGF-B gene expression in cell types other than SMCs is under the transcriptional control of the zinc finger nuclear factors, Sp1, Egr-1, and Sp3. Since the PDGF-B gene is expressed at higher levels in cultured pup rat aortic SMCs than SMCs derived from adult rats, we hypothesized that elevated PDGF-B gene expression may be due to elevated levels of Sp1, Egr-1, and Sp3 in the former cell type. Using a 32 P-labeled double-stranded oligonucleotide bearing consensus sites for this superfamily of regulatory proteins together with nuclear extracts from cultured pup or adult aortic SMCs, we observed the formation of several distinct nucleoprotein complexes, designated a-g (Fig. 1). Differences were observed in several of these complexes between the two cell subtypes. By densitometric assessment, complexes a, b, c, and d were approximately 12-, 11-, 4-, and 5-fold more abundant using extracts from pup cells as compared with adult cells, respectively.
Antibody inhibition experiments were performed to identify the protein components of these complexes. Inclusion of polyclonal antibodies directed against Sp1 in the binding reaction attenuated formation of complex a ( Fig. 2A), the most intense of these complexes. Antibodies raised against Egr-1 eliminated complex b (Fig. 2B), while antibodies recognizing Sp3 completely abolished the formation of complex c (Fig. 2C). Interestingly, the Sp1 antibody slightly attenuated the intensity of complex c while the Sp3 antibody weakly inhibited the formation of complex a (Figs. 2, A and C). This raises the possibility that Sp1 and Sp3 occupy the promoter fragment simultaneously in these cells. Indeed, the incomplete supershift obtained using the Sp1 antibody ( Fig. 2A) has been observed previously by several groups (16 -19).
Several criteria indicate that differences in Sp1, Egr-1, and Sp3 binding activity observed between pup and adult SMCs was not a consequence of unequal protein content in each FIG. 1. Greater abundance of G؉C-rich binding factors in nuclei of pup aortic SMCs than adult cells. A 32 P-labeled oligonucleotide bearing consensus elements for several zinc finger transcription factors with preference for GϩC-rich sequences ( 32 P-Oligo GC); 5Ј-GGG GGG GGC GGG GGC GGG GGC GGG GGA GG-3Ј, sense strand) was incubated with nuclear extracts from pup or adult SMCs. Binding reactions and gel electrophoresis were performed as described under "Experimental Procedures." Nucleoprotein complexes a-d are indicated by arrows. Differences in band intensity were assessed by scanning densitometry and normalized to complex f. preparation of extract. First, identical amounts of pup and adult nuclear extracts were used for each EMSA. Second, the intensity of nucleoprotein complexes e, f, and g was similar between pup and adult samples. Third, the nucleoprotein complex obtained when a 32 P-labeled oligonucleotide bearing a serum response element in the egr-1 promoter was incubated with these extracts was slightly more intense when adult samples were used (Fig. 2D).
We performed Western blot analysis to provide confirmatory evidence that Sp1, Egr-1, and Sp3 are present in greater levels in nuclear extracts from pup cells than adults since DNA bind-ing activity and levels of protein may not necessarily correlate. Findings from immunoblot analysis indicate that Sp1, Egr-1, and Sp3 levels are indeed greater in pup nuclei than adults (Fig. 3), consistent with observations from gel and supershift studies (Fig. 2). The Coomassie Blue-stained gel indicates equal protein loading (Fig. 3). Taken together, these results demonstrate that Sp1, Egr-1, and Sp3 are constitutively expressed in pup SMCs and that these levels exceed those in adult cells.
Pup Aortic SMCs Support High Basal Expression of a Reporter Gene Driven by the Zinc Finger Transcription Factor Binding Site-To demonstrate a functional consequence of the disproportionate existence of these transcription factors in the nuclei pup and adult SMCs, we mobilized the gel shift oligonucleotide upstream of CAT cDNA in pCATprom and performed transient transfection analysis using the heterologous promoter-reporter construct in each cell type. Normalized CAT activity was severalfold greater in pup SMCs than adult cells (Fig.  4). The higher levels of reporter gene expression in pup cells obtained using this construct are thus consistent with the greater abundance of factors binding the element in this cell type (Fig. 1).

Nucleotide Elements in the Proximal PDGF-B Promoter Are Required for High Basal PDGF-B Promoter-dependent Expression in Pup Aortic
SMCs-Nucleotide regulatory elements mediating basal expression of the PDGF-B gene have not yet been defined in vascular SMCs. Transient transfection analysis was performed in pup cells with a series of PDGF-B promoter fragments cloned upstream of a promoterless CAT cassette (7). CAT activity directed by construct d26, which extends 153 bp upstream of the TATA box, was comparable with expression generated from construct d77, which bears 82 bp of promoter sequence (Fig. 5). However, CAT expression produced by construct d75, which bears 19 bp of promoter sequence, was only 9% that of d77 (Fig. 5). These findings indicate that the region between the 5Ј end points of the PDGF-B fragments in d77 and d75 contain nucleotide elements crucial for basal expression in pup cells.
Sp1, Egr-1, and Sp3 Bind to the Proximal PDGF-B Promoter in Cultured Pup Aortic SMCs-We used EMSA and supershift studies to determine whether Sp1, Egr-1, and Sp3 interact with the region in the PDGF-B promoter responsible for high basal expression in pup SMCs. A 32 P-oligonucleotide spanning the Ϫ30/Ϫ13 region of the PDGF-B promoter in competition with its unlabeled cognate revealed the formation of several specific nucleoprotein complexes (Fig. 6, complexes a-d, compare lanes  2 and 4). , and Sp1 (lane 9) associate with this region of the PDGF-B promoter preferentially in pup nuclei. Interestingly, the partial supershift obtained using the Sp1 antibody alone was completely abrogated by coincubation with the Sp3 antibody (lane 10). These findings indicate that Sp1, Egr-1, and Sp3 bind to the proximal PDGF-B promoter in pup cells although, of all these factors, Sp1 associates with this region of the promoter most abundantly.

The Proximal Region of the PDGF-B Promoter Required for Basal Expression in Pup SMCs Supports Inducible Expression in Adult
Cells-Transfection of construct d77 into pup SMCs resulted in high levels of PDGF-B promoter-dependent gene expression (Fig. 7A, column 1). In comparison, reporter gene expression driven by this construct was low in adult cells (column 3). Mutation of the region bound by these factors in the PDGF-B promoter resulted in an 87% decrement in normalized CAT activity in pup cells (column 2). Interestingly, reporter gene expression directed by the mutant construct in pup cells was comparable with levels obtained using the wild-type construct in adult cells (column 3). Mutation of the zinc finger binding site (Ϫ34/Ϫ18) only slightly attenuated promoter-dependent expression in adult cells (column 4). When wild type d77 was cotransfected with a cytomegaloviral promoter-driven Sp1 expression construct in adult cells, reporter expression increased to levels comparable with those in pup cells (column 5). Overexpression of Egr-1, either alone or in combination with Sp1, also resulted in inducible PDGF-B promoter-dependent Pup SMCs were transiently transfected with a series of PDGF-B promoter-CAT constructs, and CAT activity in the lysates was determined as described under "Experimental Procedures." CAT activity was normalized to levels of growth hormone secreted into the culture medium. expression (Fig. 7B). These binding, mutational, and overexpression studies demonstrate that elevated PDGF-B expression in cultured pup SMCs is influenced by zinc finger nuclear factors binding to regulatory elements in the proximal region of the PDGF-B promoter.

DISCUSSION
In this paper, we have investigated the transcriptional mechanisms underlying the high constitutive expression of PDGF-B in SMCs isolated from the aortae of pup rats. Curiously, SMCs cultured from adult rats of the same strain express low levels of PDGF-B. EMSA and Western blot analysis revealed that levels of Sp1 and two other zinc finger transcription factors, Sp3 and Egr-1, are markedly elevated in the nuclei of pup SMCs compared with adult cells. 5Ј deletion and transient transfection analysis defined the proximal region of the PDGF-B promoter as that which mediates high basal expression of the gene in pup cells. EMSA using extracts from pup cells revealed that Sp1, Egr-1, and Sp3 bind to this region of the promoter in a specific manner. Mutation of the proximal element abolished basal PDGF-B promoter-dependent expression in these cells. Conversely, overexpression of Sp1 and/or Egr-1 in adult cells increased reporter gene expression. These studies define a critical role for the proximal region of the PDGF-B promoter in the elevated expression of the gene in pup SMCs and identifies several nuclear proteins that bind to these elements in a specific and functional manner.
While the basic structure of the rat aorta is already formed at birth, the vessel wall continues to develop within the first three months postpartum. SMCs proliferate and differentiate most actively in the first few weeks and deposit large amounts of connective tissue matrix components into the extracellular space while the artery undergoes remodeling (20). The migratory and proliferative effects of PDGF-BB on cultured aortic SMCs (2) and the chemotactic effects of PDGF-BB on SMCs in rat (21) and porcine (22) models suggest that the PDGF B-chain may play a developmental role. A thinner aortic wall, among other abnormalities, in PDGF-B null mice is consistent with a role for PDGF-B in blood vessel development (23). High constitutive PDGF-B expression in pup SMCs that decline in older mice lends support to this notion prima facie. However, Northern blot analysis indicates that PDGF-B mRNA is present only in low amounts in aortic SMCs in pup and adult rats (24). Placement of pup SMCs in culture results in a dramatic change in PDGF-B phenotype, so much so that abundant levels of PDGF-B mRNA are observed after six passages (4). Moreover, SMCs isolated from the neointima of adult rats 2 weeks after balloon catheter injury have an epitheloid morphology strongly resembling that of pup SMCs (25). Steady-state PDGF-B mRNA levels in these pup-like neointimal cells also increase with time in culture (26). In situ hybridization using rat-specific cDNA probes and an en face technique shed light on this FIG. 7. The proximal PDGF-B promoter supports elevated basal expression in pup aortic SMCs but not adult cells. A, SMCs were transiently transfected with construct d77 or d77 bearing a mutation in the site bound by Sp1, Egr-1, and Sp3, d77m (13). B, Cotransfection of d77 with Sp1-pcDNA3 and Egr-1-pcDNA3. Transfections were performed as described under "Experimental Procedures." CAT activity was normalized to levels of grown hormone secreted into the culture medium. FIG. 6. Sp1, Egr-1, and Sp3 bind to the proximal PDGF-B promoter in a specific manner. Nuclear extracts from pup rat aortic SMCs and adult SMCs were incubated with a 32 P-labeled fragment of the proximal PDGF-B promoter ( 32 P-Oligo B) and adducts separated by gel electrophoresis as described under "Experimental Procedures." For competition or antibody inhibition studies, either a 100-fold molar excess of unlabeled oligonucleotide or 2 l of the appropriate antibody was incubated with the binding mixture for 10 min prior to the addition of the 32 P-labeled probe. The specific nucleoprotein complexes a-d are indicated by arrows. apparent conundrum. It appears that only a subpopulation of SMCs can actually express PDGF-B in the aortic media. SMC cultures expressing high levels of PDGF-B after successive passages may arise from clonal expansion of the PDGF-Bexpressing SMC subpopulation (27). Cloning pup SMCs by dilute plating have generated isolates that either express or do not express PDGF-B mRNA (6). Aortic SMCs are, therefore, a heterogeneous population with diverse patterns of growth and gene expression (6). Such gross differences in PDGF-B expression between cultured pup and adult SMCs provide a valuable resource to investigate transcriptional mechanisms that contribute to cell-specific gene expression. In addition, the similarity of the PDGF-B phenotype between cultured pup SMCs and SMCs isolated from the balloon-injured rat adult artery wall (26) implicates a role for zinc finger transcription factors in the vascular response to injury.
Differences in nuclear transcription factor content have previously been suggested as a regulatory mechanism underlying cell-specific gene expression. For example, interleukin-2 expression in murine Th1 cells, but not Th2 cells, may be due to the presence of NFAT in the former cell type (28). Second, expression of Bruton's agammaglobulinemia tyrosine kinase (Btk) gene in B-cells, but not K562 cells or Jurkat T-cells, may be due to the presence of both Spi-1/PU.1 and Spi-B in B-cells (29). Third, murine surfactant protein A (SP-A) promoter-dependent expression in mouse lung epithelial (MLE-15) cells, but weakly, if at all, in HeLa, 3T3, or H441 cells, may be due to the preferential expression of thyroid transcription factor-1 (TTF-1) in the former cell type (30). Finally, MyoD, the skeletal muscle lineage control gene product that converts adult SMCs to skeletal muscle myoblasts, is unable to alter the phenotype of pup SMCs (31), suggesting that pup and adult SMCs differ in nuclear factors able to interact with MyoD (6). The present study also extends recent findings demonstrating no major differences between the content of NF-B and AP-1 in the nuclei of pup and adult cells (32). Our findings also demonstrate that overexpression of Sp1 and Egr-1 could augment PDGF-B promoter-dependent expression in rat SMCs and that these effects were not additive under these conditions. Gel shift studies indicate that in addition to Sp1 and Egr-1, Sp3 and other factors, yet to be defined, bind specifically to the proximal PDGF-B promoter (Fig. 6). While overexpression studies are helpful in determining the functional importance of transcription factors, simultaneous overexpression of multiple factors with a reporter may not necessarily approximate the expression pattern of the authentic gene. This process is likely to be influenced by the nature, concentration, stoichiometry, and even the order of assembly of nuclear factors on the promoter.
While the present findings indicate that the proximal region of the PDGF-B promoter is required for high basal PDGF-B promoter-dependent expression in pup cells and that this region is bound by a large number of nuclear factors, elevated PDGF-B expression may not be mediated by these trans-acting factors alone. For example, basal and inducible PDGF-A transcription is, like PDGF-B, dependent upon specific interactions of Sp1, Sp3, and Egr-1 with the proximal promoter (33). Unlike PDGF-B, however, steady-state PDGF-A mRNA levels are reportedly similar in pup and adult SMCs but do increase with time in culture (4,6). This may result from differences in the methylation state of the authentic promoter, or combinatorial interactions between transcription factors over promoter elements. Methylation of the herpes simplex virus thymidine kinase promoter results in loss of DNase I hypersensitivity and inhibition of transcription (34). Prolactin promoter-dependent gene expression in pituitary tumor cells is inversely correlated with CpG methylation of the promoter and the ability of tran-scription factors to bind (35). The GϩC-rich nature of the PDGF-A promoter adds support to this possibility (36). Second, transcription factors undergo ordered cooperative interactions over promoter elements to form stereospecific enhancer complexes that augment rates of transcription (37). Sp1 bends DNA upon promoter binding (38) and interacts synergistically with YYI (39), Ets-1 (40), HIV Tat (41), GATA-1 (42,43), and p53 (44). Differences in the nature or content of other regulatory factors may, therefore, influence this highly selective process and contribute to the preferential expression of PDGF-B in cultured pup SMCs.


s2fieldsofstudy: Biology Medicine

text:
Oxaliplatin in practice.

Oxaliplatin, a new third-generation platinum complex, is active in the treatment of colorectal and advanced ovarian cancers, both as monotherapy and in combination therapy. It has demonstrated a very good safety profile, characterized by low haematotoxicity, and moderate and manageable gastrointestinal toxicity. No significant renal or ototoxicities have been observed. Oxaliplatin induces a peripheral sensory neuropathy which is characterized by distal and perioral dysaesthesia, and is induced or exacerbated by the cold; in general, it is regressive between cycles of treatment. This dose-limiting toxicity is cumulative, but reversible within a few months of discontinuation of treatment in the majority of cases. In a cohort study of 490 patients with advanced colorectal cancer included in an extended access programme, more than 2700 cycles of oxaliplatin plus 5-fluorouracil (5-FU) were administered. The overall safety profile of oxaliplatin was shown to be very favourable. Oxaliplatin and cisplatin, each in combination with cyclophosphamide, have a similar efficacy in the treatment of advanced ovarian cancer, but oxaliplatin was better tolerated than cisplatin in terms of haematological, gastrointestinal, neurosensory and renal toxicities. The safety profile of oxaliplatin makes it an ideal candidate for combination therapy.

Oxaliplatin was introduced into clinical trials by Mathe and colleagues in 1986, and has been approved in France for the treatment of advanced colorectal cancer, both as monotherapy and in combination with 5-fluorouracil (5-FU)/folinic acid (FA), for more than a year. To date, more than 2000 patients have received oxaliplatin in clinical trials, 1400 of these for the treatment of metastatic colorectal cancer. All of these studies have confirmed the efficacy and favourable toxicity and safety profiles of oxaliplatin.
Oxaliplatin as monotherapy has also shown activity in pretreated ovarian cancer patients (Misset et al, 1991). The lack of cross-resistance with cisplatin and carboplatin has been demonstrated in human ovarian cancer cell lines, both in vitro and in vivo (Pendyala et al, 1993;Alvarez et al, 1994). This activity profile, together with an excellent tolerability, provides the rationale for the use of oxaliplatin in combination with cisplatin in pretreated ovarian cancer patients. The combinations of oxaliplatin-cisplatin and oxaliplatin-cisplatin-epirubicin-ifosmamide have been shown to be active in such patients (Soulie et al, 1996(Soulie et al, , 1997.
Oxaliplatin is a non-conventional platinum compound that differs from cisplatin in its lack of nephrotoxicity and from carboplatin in its association with only limited haematological toxicity. The most frequent acute side-effect of oxaliplatin is a transient peripheral neuropathy, manifesting as parasethesia and dysaesthesia in the extremities, which is triggered or enhanced by exposure to cold. These are discussed below.

TOXICITY PROFILE Haematotoxicity
In a large phase I study, 44 patients with advanced cancer received 116 courses of oxaliplatin, with dose escalation from 45 to 200 mg m-2 (Extra et al, 1990). Most patients had previously received chemotherapy. Moderate haematological toxicity was observed. Thrombocytopenia was dose related and did not occur at doses of oxaliplatin less than 90 mg m-2; however, it did occur in 13% of patients receiving doses of 135-150 mg m-2, and 28.5% of those treated with 175-200 mg m-2 exhibited a decreased platelet count that did not exceed World Health Organization (WHO) grade 2. Similarly, only grade 1 or 2 neutropenia was observed, and haemoglobulin levels generally remained unchanged. Table 1 summarizes the incidence of grade 3 and 4 haematotoxicity as a result of the administration of oxaliplatin in combination with 5-FU/FA (Levi et al, 1992;de Gramont et al, 1997). The 39% grade 3 and 4 neutropenia observed in the Folfox 2 study was manageable, and was a result of 5-FU. It could be avoided in most patients as it almost always occurred after 5-FU dose escalation, and did not recur after further dose adjustment. No haematopoietic growth factors were used.
Thus, oxaliplatin in combination with 5-FU/FA does not increase the haematological toxicity. Indeed, the profile resulting from this treatment combination is almost entirely the result of the 5-FU/FA component of the treatment.
Renal toxicity Unlike cisplatin, oxaliplatin does not appear to be nephrotoxic, and its administration does not require any specific nephroprotective measures (e.g. hyperhydration). The pharmacokinetic behaviour of oxaliplatin has been evaluated in patients with normal and impaired kidney function (creatinine clearance value, < 60 ml min-') (Massari et al, 1994;Raymond et al, 1998). Results showed that, after a 2-h infusion of oxaliplatin, no differences were observed between the plasma concentration of the drug in patients with renal impairment and in those with normal renal function, suggesting that dose modification was not required in patients with impaired renal function. Forty-nine patients with impaired renal function have been treated with full-dose oxaliplatin, either as monotherapy or in combination, without evidence of increased nephrotoxicity (Massari et al, 1996). Gastrointestinal toxicity Nausea and/or vomiting were observed in the majority of patients, and it did not seem to increase with the addition of 5-FU (Table 2). Severe nausea and vomiting were seen in 10% of patients treated with oxaliplatin as monotherapy and in 22% of those treated with oxaliplatin in combination with 5-FU. These symptoms can usually be controlled with supportive measures, including anti-5HT3 medications and loperamide. Diarrhoea appears to be more frequent and severe with longer infusion schedules. Severe diarrhoea occurs in approximately 4% of patients who have received oxaliplatin monotherapy compared with 25.3% of patients who have received oxaliplatin plus 5-FU. The incidence of severe mucositis is dependent on the 5-FU regimen used in the studies.

Neurotoxicity
Acute neurotoxicity associated with oxaliplatin is common (85-95% of patients) but usually not dose-limiting, occurring within hours of treatment and regressing between treatments. Typical symptoms have included paraesthesias, usually presenting Cumulative dose (mg m-2) Figure 1 The cumulative incidence rate of grade 3 (specific scale) neuropathy in 682 patients receiving oxaliplatin as monotherapy or in combination therapy as cold-related dysaesthesias, which were seldom painful or associated with cramps. The majority of these toxicities lasted for 7 days or less, and resolved between treatments. A sporadic, sometimes sudden, and self-limiting laryngopharyngeal dysaesthesia is thought to result from decreased sensitivity of the larynx and pharynx, which causes a feeling of difficulty in breathing or swallowing. Symptoms have resolved spontaneously within hours of onset. Symptoms tended to last longer with successive cycles. Neurological toxicity scales currently available are generally insufficient for grading the characteristics of the dysaesthesia associated with oxaliplatin use. Consequently, a grading system was developed by Levi and co-workers that takes into account both intensity and duration of symptoms related to oxaliplatininduced paraesthesia/dysaesthesia (Caussanel et al, 1990). This is shown in Table 3, in which it is compared with the WHO grading system for neurotoxicity. Table 2 Gastrointestinal toxicity found with oxaliplatin as monotherapy and in combination with 5-FU/FA (Massari et al, 1996) (4)  16 (5)  5 (1) 10 (2) 17 (5) 6 (1) 11 (6) < 1 (< 1) < 1 (< 1) 3 (< 1) 3 (1) < 1 (< 1) A recent safety evaluation in 682 patients, who received either oxaliplatin alone or in combination with 5-FU, indicated that, at a mean cumulative dose of 900 mg m-2, 12% of patients experienced grade 3 neurotoxicity, which presented as fine movement disturbances and moderate sensory ataxia (Figure 1) (de Gramont et al, 1997). Further evaluation has shown neuropathy grade 3/4 (Levi's scale) to occur with oxaliplatin in 75% of patients after 12 cycles and administration of a cumulative dose of 1560 mg m-2.
However, this cumulative sensory neuropathy is generally reversible after discontinuation of treatment, and disappears entirely at 6-8 months in 42% of patients (Table 4). The incidence of cumulative neurotoxicity was similar for oxaliplatin monotherapy and the combination of oxaliplatin plus 5-FU ± FA. Little or no ototoxicity has been observed.

SAFETY PROFILE Overall safety
In the phase IV Extended Access Programme, patients were treated with oxaliplatin, either as monotherapy or in combination with 5-FU/FA. Safety data from 490 patients and 2702 cycles of treatment have now been evaluated. Some of the patients were heavily pretreated with oxaliplatin, being the fifth line of treatment, with a median cumulative dose of 600 mg m-2 (range 70-2300 mg m-2). However, the overall safety profile of oxaliplatin in this population of patients proved to be very favourable (Table 5) and confirmed the clinical trial results.
One phase III trial, in which the combination of oxaliplatin plus cyclophosphamide was compared with the combination of cisplatin plus cyclophosphamide, has been completed in patients with advanced ovarian cancer (Misset et al, 1997). The primary objective of this multicentre trial was to evaluate the safety profile of each treatment, and the secondary objectives were to compare efficacy, progression-free survival and overall survival. Patients (n = 182) were randomized to receive cisplatin 100 mg m-2 i.v. for 1 h every 3 weeks or oxaliplatin 130 mg m-2 i.v. for 2 h every 3 weeks, for six cycles of treatment. Both groups also received cyclophosphamide 1000 mg m-2 i.v. for 2 h every 3 weeks.
An interim analysis of the safety data was performed and the results showed that the treatment in both arms of the study was acceptable, with over 450 cycles of treatment being administered in each group. It was found that almost 90% of the planned dose of oxaliplatin could be given. The cisplatin dose was reduced to 85.7% because of haematotoxicity. In addition, the dose of cyclophosphamide was reduced further in this group, to 85% compared with 90.5% in the group receiving oxaliplatin.
Haematotoxicity of grade 3/4 caused a delay in twice the number of cisplatin treatment cycles compared with oxaliplatin (141 compared with 70 for all grades of haematotoxicity: P = 0.001). Gastrointestinal toxicity was also lower in the group of patients receiving oxaliplatin, with high-grade nausea and vomiting occurring in a significantly smaller proportion of patients (oxaliplatin 7.9% compared with cisplatin 25.9%: P = 0.001).
When the levels of neurosensory toxicity were investigated, it was found that during the early stages of the trial, low-grade toxicity occurred much more frequently in the group of patients treated with oxaliplatin. By the end of the therapeutic programme, however, there was a complete absence of the anticipated grade 3 toxicity in this patient group, while cumulative neurotoxicity was Oxaliplatin in practice 7 found with cisplatin. Thus, in clinical practice, it should be possible to treat patients with the recommended full programme of six cycles of oxaliplatin. Renal toxicity was shown in 9% and auditory toxicity in 4.5% of patients treated with cisplatin, but neither toxicity was seen in patients receiving oxaliplatin. Asthenia was also found more frequently in patients treated with cisplatin (oxaliplatin 9.2% compared with cisplatin 20.2%; P = 0.04).
The time-related parameters of this study are shown in Table 6. The overall survival parameters of the two groups are very similar at present, but the final analysis has yet to be completed.
This study has shown oxaliplatin to have a better safety profile than cisplatin, in terms of haematological, gastrointestinal, neurosensory and renal toxicities in the treatment of patients with advanced ovarian cancer. No significant difference has yet been observed between the two treatments in objective response rate, time to progression and overall survival.

CONCLUSIONS
Oxaliplatin is a well-tolerated anticancer drug and a good candidate for combination therapy. The neurotoxicity that is characteristic of the drug is acute and dose related, but is generally reversible on discontinuation of treatment and can be managed satisfactorily in clinical practice.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Phosphorylation of Vitronectin by Casein Kinase II

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain ∼3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a K m of 0.5–2 μm (lower than the Vn concentration in blood, 3–6 μm), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the αvβ3 Vn receptors.

coprotein in the extracellular matrix of various cells and in circulating blood (2)(3)(4). It promotes cell attachment, spreading, and migration (5)(6)(7)(8) through an RGD sequence that is known to be recognized by the integrins (9,10). In addition, Vn was shown to participate in the regulation of the complement function (11)(12)(13)(14)(15) and in the control of plasminogen activation, by stabilizing the inhibitory conformation of the inhibitor-1 of plasminogen activators, leading to an arrest of the production of plasmin from plasminogen (16 -22).
We have previously shown that Vn is the major protein substrate in serum for protein kinase A (PKA), which is also found as an ectoenzyme in several types of blood cells (23,24). 2 PKA was shown to be released into the blood fluid from platelets upon their activation with thrombin (24,25). The distinct selectivity of this Vn phosphorylation by PKA and the finding that this kinase is released from the platelets in response to a key physiological (thrombin) stimulus in hemostasis suggested to us that this kinase-driven covalent modification may have a physiological regulatory importance. Support for this notion came from reconstitution experiments using Vn isolated from human plasma and a pure catalytic subunit of PKA. These experiments revealed that under the conditions existing at the locus of a hemostatic event, the phosphorylation of Vn by PKA may well take place in vivo (26 -28). Moreover, we found that following this PKA phosphorylation, there is a conformational change in Vn (which was demonstrated by circular dichroism) that results in a significant reduction in the binding of Vn to the inhibitor-1 of plasminogen activators (29) and, consequently, in the modulation of an important physiological function of Vn.
The specificity (Ser 378 only) and stoichiometry of the PKA phosphorylation of plasma Vn as purified from human blood (24,26,28,30,31) suggested to us that in vivo, Vn is most likely phosphorylated also by other protein kinase(s) besides PKA, because plasma Vn was found to contain ϳ3 mol of covalently bound phosphate per mol of Vn (30,31). Indeed, we have recently shown that in vitro Vn is also a substrate for PKC, which phosphorylates it at Ser 362 (again with a stoichiometry of ϳ0.7 mol/mol), and subsequently attenuates the cleavage of Vn by plasmin (32). However, if we take together these PKA and PKC phosphorylations, they are not sufficient to account for the stoichiometry of ϳ3 mol of phosphate per mol of Vn, which was determined prior to the in vitro phosphorylation of the vacant phosphorylation sites of Vn. Therefore, we assumed that in vivo, there might still be an additional kinase (and maybe more) that might phosphorylate Vn. In fact, this possibility seemed plausible on the basis of our earlier findings that (a) upon fractionation of human serum on an anion-exchange column, we identified three peaks of protein kinase activity one of which phosphorylates phosvitin (33), and (b) that the phosphorylation of Vn by platelet ectokinases was not completely inhibited by the PKA and PKC inhibitors PKI and calphostin C. 3 A plausible candidate for this purpose seemed to be CKII, because a protein kinase activity with a CKII-like specificity was found in serum (33) and, as an ectoenzyme, on epithelial cells (34,35), neutrophils (36,37), endothelial cells (38,39), and platelets (40,41). 3 Here, we report the biochemical characterization of the CKII phosphorylation of Vn, focusing on its possible physiological significance. Plasma Vn is shown to be a substrate for CKII and to phosphorylate Thr 50 and Thr 57 (i.e. amino acid residues vicinal to the cell-adhesion RGD site of Vn (positions [45][46][47]). The functional consequences (and thus the possible physiological relevance) of this CKII phosphorylation is demonstrated by comparing wt-Vn to the CKII phosphorylated wt-Vn, as well as the two double mutants: T50E/T57E (a mutant representing an analog of a fully phosphorylated Vn at Thr 50 and Thr 57 ) and T50A/T57A (a mutant representing an analog of a nonphosphorylated Vn at these two positions). The adhesion of bovine aorta endothelial cells (BAECs) onto the CKII phosphorylated wt-Vn or onto its T50E/T57E mutant is shown to be enhanced (via an ␣ v ␤ 3 integrin) in comparison to their adhesion onto the nonphosphorylated wt-Vn, as well as onto its T50A/T57A mutant.
Other Proteins and Enzymes-Vn was purified from freshly frozen human plasma as described previously (12), with the modifications used routinely in our laboratory (25,27). The catalytic subunit of PKA was purified according to the method described by Beavo et al. (42).
Thrombin Cleavage of Vn (28,43)-The reaction mixture (50 l), contained the following components at the final concentrations given in parentheses: CKII phosphorylated Vn (50 g/ml), thrombin (30 IU/ml), and Hepes buffer (30 mM, pH 7.5). The reaction was allowed to proceed for 30 min at 37°C and was arrested by the adding 12 l of 5ϫ Laemmli's sample buffer, boiling for 3 min, and then subjecting it to SDS-PAGE.
Increasing the Stoichiometry of the CKII Phosphorylation of wt-Vn by Activation of the Kinase-Samples of wt-Vn or T50A/T57A-Vn were phosphorylated in vitro by CKII in the following reaction mixture: CKII (2 g/ml), glycerol (5%), NaCl (50 mM), ␤-mercaptoethanol (5 mM), magnesium chloride (10 mM), [␥-32 P]ATP (10 M, 6 -30 Ci/mmol), Hepes buffer (30 mM), pH 7.5, in the absence or presence of 15 g/ml poly-L-lysine. The reaction was allowed to proceed at 22°C for 30 min, arrested by boiling for 5 min in Laemmli's sample buffer, then loaded on SDSpolyacrylamide gels (10% acrylamide). The gel was dried and exposed to autoradiography, and the radioactive phosphorylated Vn bands were excised from the dried gel and counted in a scintillation counter. The stoichiometry of phosphorylation was calculated on the basis of the specific radioactivity of [␥-32 P]ATP.
Preparation of Phosphorylated wt-Vn and Nonphosphorylated wt-Vn for Cell Adhesion Assays-The reaction mixture was prepared as described above except for the fact that [␥-32 P]ATP was replaced by nonradiolabeled ATP. For the preparation of nonphosphorylated wt-Vn, ATP was substituted by AMP-PCP. All other constituents were the same. The reaction was allowed to proceed at 22°C for 30 min; thereafter, the reaction mixture was serially diluted and applied onto 48-well plates for the cell adhesion assay.
Acid Cleavage of Vn-Vn was phosphorylated by CKII as described above and subjected to SDS-PAGE (10% acrylamide). The gel was electroblotted onto a nitrocellulose membrane, and the membrane was then washed with distilled water and exposed to autoradiography. The Vn bands were localized in the membrane by superimposing the membrane onto the autoradiogram, excised, and transferred into a microtube containing 100 l of 70% formic acid. After incubation for 12 h at 37°C, the extract was diluted with double distilled water 7-10 fold, concentrated in a Speedvac, dissolved in Laemmli's sample buffer, boiled (3 min), and subjected to SDS-PAGE.
Phosphoaminoacid Analysis of Phosphorylated Vn-Phosphoaminoacid analysis was carried out as described previously (44).
Purification and Sequencing of a Tryptic Peptide from Vn Harboring the CKII Phosphorylation Site-A sample of Vn (2 nmol), was heated to 56°C for 15 min to allow maximal CKII phosphorylation (cf. Fig. 1) and then chilled on ice. Phosphorylation by CKII (0.5 g) was allowed to proceed for 60 min at 22°C in a reaction mixture (200 l) containing magnesium acetate (10 mM), [␥-32 P]ATP (10 M, 6 Ci/mmol), and Hepes (50 mM, pH 7.5). The reaction was arrested by an addition of a 50-fold excess of nonradioactive ATP. Then, Vn was reduced (15 min with 10 mM dithiothreitol, pH 7.5, at 56°C), alkylated with 30 mM iodacetamide (for 1 h in the dark), and precipitated by ammonium sulfate (70%) to separate the protein from the excess radioactive ATP; it was then extensively dialyzed overnight at 4°C against 50 mM Hepes, pH 7.4, and digested with trypsin (sequencing grade, 5 g) at 36°C for 6 h. The material was diluted 1:10 with double distilled water containing 0.1% trifluoroacetic acid, applied to a C 8 reverse phase-high pressure liquid chromatography column, and eluted by a linear gradient of acetonitrile (0 -80%) for 120 min. The flow rate was 0.33 ml/min. The absorbance peaks (214 nm) were collected manually and counted on a scintillation counter. The peaks that contained the highest amount of radioactivity were selected for amino acid sequence analysis.
Tissue Cultures-BAECs were grown in tissue culture dishes in DMEM (low glucose; 1 g/liter) supplemented with 10% (v/v) calf serum (Life Technologies, Inc.). NIH-3T3 cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated calf serum (HyClone). The cells growing was carried out in an incubator (37°C) with an atmosphere containing 5% CO 2 . The Sf-9 and High-5 insect cells were maintained in Grace's insect medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal bovine serum. For the expression of recombinant Vns, a serum-free medium (Sf-900 II, Life Technologies, Inc.) was used. All media were supplemented with 50 g/ml Gentamicin and 12.5 g/ml Fungizone (Life Technologies, Inc.).
Adsorption of Vns onto the Plastic Surface of Microtiter Plates for Enzyme-linked Immunosorbent Assay-Vn solutions serially diluted in PBS were added to 96-well microtiter plates for 2 h at 22°C, and the residual nonspecific adsorption sites were blocked with 0.3% bovine serum albumin in PBS for 1 h. Bound Vn was determined with specific rabbit anti-human Vn polyclonal antibodies and goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1 h at 22°C). After extensive washings with PBS containing 0.01% Tween 20, the chromogenic substrate 2,2Ј-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) was added. The reaction was monitored in an enzyme-linked immunosorbent assay reader at 620 nM.
Cell Adhesion Assay-Serial dilutions of Vns were added to 24-well plates (250 l) for 1.5 h at 22°C to allow coating of the plates. Thereafter, the solutions were aspirated, and 0.5 ml of DMEM containing 1 mg/ml hemoglobin was added for 30 min at 37°C. Confluent BAECs or NIH-3T3 cells plated on 10-cm plates were labeled with 30 Ci [ 35 S]methionine for 3-4 h at 37°C. The cells were then trypsinized and collected into DMEM containing 0.2 mg/ml soybean trypsin inhibitor to arrest trypsin action. The cells were then centrifuged (5 min at 1200 ϫ g) and resuspended in DMEM containing 1 mg/ml hemoglobin adjusting 3 I. Schvartz, T. Kreizman, and S. Shaltiel, unpublished results. the cell concentration to 10 6 cells/ml. Cell suspensions (250 l) were added to each coated well for 30 min at 37°C. The cells were washed three times with 0.5 ml of PBS, and the adhered cells were treated with 0.5 ml of 1% Triton X-100 in PBS for 5 min. Samples of 0.4 ml were transferred into scintillation vials for counting. The quantitation of cell adhesion is reported as the residual radioactivity (a mean of triplicates in cpm) of the cells to be tested, after their extensive washing (three times with 0.5 ml of PBS). This comparison was convenient and valid, because each assay was carried out with an identical volume of cell suspension and an identical number of cells (ϳ700,000 cpm/250,000 cells). Cell adhesion assays of the nonphosphorylated wt-Vn and the CKII phosphorylated wt-Vn were performed in 48-well plates. All of the components and treatments of the assay were scaled down accordingly.
Inhibition of BAEC Adhesion by Function-inhibiting Monoclonal Antibodies-The monoclonal antibodies used were as follows: P1F6, directed against the integrin receptor ␣ v ␤ 5 ; LM609, directed against the integrin receptor ␣ v ␤ 3 ; and HA, directed against hemagglutinin as control. Plates (24 wells) were coated with a 5 g/ml concentration of the Vn to be assayed (250 l) for 1.5 h 22°C, and then the nonspecific adsorption sites were blocked with 0.5 ml of DMEM containing 1 mg/ml hemoglobin (30 min. at 37°C). The cells were treated as described above (under "Cell Adhesion Assay") and resuspended in DMEM containing 1 mg/ml hemoglobin to yield a concentration of 10 5 cells/ml. Before starting the cell adhesion assay, the cells were preincubated with different amounts of monoclonal antibodies (with gentle shaking) as indicated in the legend to Fig. 8 (30 min at 22°C). Thereafter, the cells were washed once with 10 ml of DMEM containing 1 mg/ml hemoglobin and resuspended to yield a concentration of 10 5 cells/ml. An aliquot of this cell suspension (250 l) was added to the Vn-coated wells, and the adhesion assay was allowed to proceed as described above.
FACS Analysis of ␣ v ␤ 3 and ␣ v ␤ 5 Expression in BAECs-Confluent BAECs were grown on 10-cm plates and then trypsinized and suspended in DMEM containing 0.2 mg/ml soybean trypsin inhibitor to arrest trypsin action. The cells were then centrifuged (5 min at 1200 ϫ g) and resuspended in DMEM containing 1 mg/ml hemoglobin to a concentration of 5 ϫ 10 5 in 100 l. The cells were incubated with anti-␣ v ␤ 3 or with anti-␣ v ␤ 5 monoclonal antibodies (final concentration, 4 g/100 l) for 1 h on ice with occasional agitation. They were then washed three times with 1 ml of a PBS buffer containing 1% bovine serum albumin and 0.02% sodium azide using a cooled microcentrifuge (4°C). After the last wash, the cells were resuspended in 100 l of the above mentioned buffer, supplemented with goat anti-mouse IgG that was conjugated with fluorescein isothiocyanate (final concentration, 5 g/100 l). The cells were allowed to bind the antibodies for 1 h (on ice) with occasional agitation and then washed as above and resuspended in 0.5 ml of PBS buffer (described above) for FACS analysis in a Becton Dickinson FACScan (530 filter). For each antibody, 5000 cells were analyzed. Control cells were incubated with the secondary antibody only.
Cell Spreading Assays-Samples (20 g) of wild-type or mutant Vn were diluted in PBS to a final volume of 2 ml and added to 6-well plates (Nunc) for 2 h at 22°C. After coating, the solutions were aspirated, and 0.5 ml of DMEM containing 1 mg/ml hemoglobin was added for 30 min at 37°C to block the residual nonspecific adsorption sites. BAECs or NIH-3T3 cells (70% confluent) plated on a 10-cm plate were serumstarved for 16 h; thereafter, the cells were trypsinized and brought as described above to a concentration of 10 5 cells/ml. A sample (4 ml) of the cell suspension was added to each coated well. After 8 min in an incubator, unattached cells were gently aspirated, and attached cells were supplemented with 4 ml of warmed DMEM (37°C) containing 1 mg/ml hemoglobin. Spreading of cells was monitored on Zeizz Axiovert 35 microscope containing a heating unit plate TRZ 3700, calibrated at 37°C. Pictures were taken at the times indicated in Fig. 7.
Preparation of Recombinant Wild-type Vn for Expression in Insect Cells-Human Vn cDNA was generously provided by Dr. Erkki Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA) (45). This clone includes no 5Ј-untranslated DNA and is missing the first three amino acids; therefore, the transcription initiation sequence (46) and the first three amino acids (Met-Ala-Pro) were added to the clone by PCR. The sense oligonucleotide sequence used is 5Ј-GGGAGCTC-GCCACCATGGCACCCCTGAGACCCCTTCTCATA-3Ј.
The antisense oligonucleotide is located downstream the SacI site at nucleotide 445. The PCR product was digested with SacI and ligated to SacI-digested pRSET-wt-Vn. The construct obtained by PCR amplification was sequenced past the SacI sites in order to confirm a correct amplification before further subcloning into the PVL1393 transfer vector (PharMingen).
Mutations of the CKII Phosphorylation Sites in Vn-All mutants were obtained by PCR amplification and insertion into an ACCI site that was especially designed by a silent mutation near Thr 57 as follows: The two amino acids following Thr 57 are Val 58 and Tyr 59 , which are encoded by GTCTAT. This sequence was changed to GTCTAC, which still encodes for Val and Tyr but also creates an ACCI site. The fragments were designed to contain also the mutated threonines using the oligonucleotide sequences given below. The sense oligonucleotide used to prime all mutants was 5Ј-TAAGAATCCGAGCTCGCCACC-3Ј. The antisense oligonucleotides that were used to generate each one of the mutations are as follows: for the T50A mutant, 5Ј-GTCGTAGACCGT-GTACTCATCCTCCGGCATAGCGAACACATC-3Ј; for T50E mutant, 5Ј-GTCGTAGACCGTGTACTCATCCTCCGGCATCTCGAACACATC-3Ј; for the T50A/T57A double mutant, 5Ј-GTCGTAGACCGCGTACTCATC-CTCCGGCATAGCGAACACATC-3Ј; and for the T50E/T57E double mutant, 5Ј-GTCGTAGACCTCGTACTCATCCTCCGGCATCTCGAACAC-ATC-3Ј. All PCR amplified fragments were ligated into the BamHI-ACCI site of Vn cDNA-pRSET. The construction of the mutations deleted a fragment of 588 base pairs, which was latter added to all mutants by PCR amplification of the missing fragment. The pair of oligonucleotides that were used for that purpose were the sense oligonucleotide 5Ј-TACGAG-GTCTACGACGATGGC-3Ј and the antisense oligonucleotide 5Ј-CCT-GACTGGGCTGGTGCTGGA-3Ј. The PCR-amplified fragment digested with ACCI and ligated into the ACCI-digested Vn mutant pRSET, which was missing this fragment. All amplified PCR products were sequenced after subcloning in order to ascertain correct amplifications, and they were then ligated into the PVL1393 transfer vector (PharMingen).
Forced Recombination into the Viral DNA and Plaque Purification-Each transfer vector containing Vn cDNA was cotransfected along with linear viral DNA into Sf-9 cells as described by PharMingen. Isolated plaques (purified by agarose overlays) as described in Ref. 47 were analyzed and amplified for further use.
Expression of Vns in High 5 Insect Cells-High titer virus stocks obtained from purified plaques were used to infect High 5 insect cells. Infected cells were grown in serum-free medium (Sf-900 II from Life Technologies, Inc.) in order to eliminate contamination of the recombinant Vn with bovine Vn from fetal bovine serum. The expressed Vns, which were secreted into the medium by their own signal, were collected 72 h after the infection.

Stoichiometry of the CKII Phosphorylation of Vn-When
Vn (purified from human plasma) was subjected to phosphorylation by CKII in the presence of [␥-32 P]ATP, an incorporation of 32 P occurred with a stoichiometry of ϳ0.1 mol of phosphate per mol of Vn (Fig. 1A). If Vn was heat-treated (15 min at 56°C) prior to the phosphorylation (a treatment that opens up the Vn molecule and favors its multimerization (48)), the stoichiometry of phosphorylation was increased but reached a plateau at ϳ0.35 mol/mol (Fig. 1A). As seen in Fig. 1B, under both conditions, the phosphorylation was targeted mainly on Thr residues, suggesting that the increased stoichiometry following heat treatment may be due to an increased exposure of the target Thr residues involved. It should be noted that although there is an apparent increase in phosphoserine content in Fig.  1B, the ratio between Thr and Ser phosphorylation before and after heat treatment is essentially the same (Ͻ9%, as judged by densitometric scanning). On the other hand, the fact that even after heat treatment, CKII phosphorylated the plasma-purified Vn only up to ϳ0.35 mol/mol indicated that some of the Vn molecules in plasma may already be prephosphorylated at these Thr residues, and they probably constitute part of the phosphate content of plasma Vn (ϳ3 mol/mol) determined by Tollefsen and co-workers (30). Interestingly, the preheated Vn appears to be a better substrate for CKII ( Fig. 2A), as indicated by the kinetic analysis of the CKII phosphorylation of Vn (Fig.  2B), which showed that the heat treatment results in a 4-fold decrease of the K m (from 2 to 0.5 M; Fig. 2B).
Localization of the CKII Phosphorylation Sites in Vn-Unlike PKA, which in the absence of heparin, preferentially phosphorylates the one-chain form of Vn (Fig. 3, A and B, lane 1), CKII phosphorylates both the one-chain and the two-chain forms of Vn (Vn 75 , and Vn 65ϩ10 respectively) (Fig. 3A, lane 2), indicating that the CKII phosphorylation site(s) is located between residues Asp 1 and Arg 379 (the Arg 379 -Ala 380 bond is the endogenous cleavage site that gives rise to Vn 65ϩ10 ). Exposure of the CKII-phosphorylated Vn to thrombin, which cleaves Vn specifically at the Arg 305 -Thr 306 bond (28) to yield a 57-kDa clipped Vn (Fig. 3, A and B, lane 3), leaves the 32 P label in the Vn 57 band, implying that the CKII phosphorylation site(s) in Vn, are located within the Asp 1 to Arg 305 segment of Vn. Finally, upon cleavage of CKII phosphorylated Vn at its acidlabile bond (Asp 217 -Pro 218 ) leaves essentially all the label in the 40 kDa fragment (the large acid-cleavage-fragment of Vn containing the N terminus of intact Vn (49) (Fig. 3A, lane 4). In view of these results, we presumed that the CKII phosphorylation site(s) in Vn is located between residues Asp 1 -Asp 217 (Fig.  3B, lane 4).
Sequencing Tryptic Peptides of CKII-phosphorylated Vn-The commonly accepted consensus sequence for CKII phosphorylation is Ser/Thr-X-X-Glu/Asp (50). However, the presence of additional Asp or Glu residues at positions Ϫ3, ϩ1, ϩ2, ϩ4, ϩ5, or ϩ7 (relative to the Ser or Thr residue to be phosphorylated) improves the phosphorylation efficacy (V max /K m ) (51,52). In fact, most of the physiological substrates of CKII have at least one additional acidic amino acid residue in addition to the ones in the consensus sequence given above (50). Phosphoamino acid analysis of CKII-labeled Vn showed that this kinase phosphorylates Vn on Thr residues (Fig. 1B). There are four Thr residues in the 40-kDa fragment of Vn mentioned above that meet with the consensus sequence requirements of CKII targets: Thr 44 , Thr 50 , Thr 57 , and Thr 69 . Of these, Thr 50 and Thr 57 should be preferred targets for phosphorylation because they contain additional acidic residues in the preferred positions mentioned above.
In an attempt to identify the exact CKII phosphorylation site(s) in Vn, we digested the CKII-phosphorylated 32 P-labeled Vn with trypsin, and the resulting mixture of tryptic peptides was applied on an reverse phase-high pressure liquid chromatography C8 column, monitoring the absorbance of the peaks (at 214 nm) and their radioactivity. The emerging peak that had the highest radioactivity (57% of total) was analyzed and found to start with the sequence 46 GDVFTMPEDE 55 (data not shown), fitting the tryptic fragment 46 GDVFTMPEDEYTVYD-DGEEK 65 . On the basis of this finding and the results summarized in Fig. 3, we concluded that the CKII phosphorylation sites in Vn are most likely Thr 50 or Thr 57 , or both. However, in view of the low yields and poor resolutions obtained when attempting to further degrade and purify the labeled peptides, we could not establish unequivocally which of these three possibilities is correct.
Identifying the CKII Phosphorylation Site(s) in Vn by Singlesite and Double-site Mutations-In order to determine the site(s) of the CKII-phosphorylation in Vn, we expressed wt-Vn and other r-Vns in a baculovirus expression system and obtained a set of Vn mutants in which each one of the two Thr residues mentioned above was replaced either by Ala or Glu. This resulted in the following r-Vn derivatives: T50A, T50E, T57A, and T57E (Fig. 4A), which we subjected to phosphorylation by CKII. As seen in Fig. 4, A and B, under the conditions of our experiment, the CKII phosphorylation was not completely abolished in any one of the four Vn mutants (T50A, T50E, T57A, and T57E). Therefore, we presumed that both threonines are probably targets for phosphorylation. To confirm this hypothesis, we constructed two double mutants in which both threonines were mutated either to Ala (T50A/T57A) or to Glu (T50E/T57E). Indeed, these two mutants were not phosphorylated by CKII (Fig. 4, A and B), demonstrating that these (and only these) threonines are the CKII phosphorylation sites in Vn. Under the conditions of the experiment depicted in Fig. 4, the incorporation of 32 P into wt-Vn was double, compared with each of the single mutations, T50A, T50E and T57A, which would be in line with the fact that the CKII phosphorylation occurs on both Thr 50 and Thr 57 (Fig. 4, A and B). However, the T57E mutant incorporated almost double the expected amount of 32 P into the remaining vacant site, Thr 50 . The reason for the enhanced incorporation of 32 P in the T57E mutant is apparently due to the fact that upon insertion of an acidic residue (a Glu residue at position ϩ7 relative to Thr 50 ), one obtains an improved CKII phosphorylation site in Thr 50 , as established in the systematic specificity studies of Pinna and co-workers (51,52).
The CKII Phosphorylation of Vn Enhances Its Ability to Promote Cell Adhesion and Spreading-To assess the effect of the CKII phosphorylation on Vn function, and thus on its possible physiological importance, we needed to compare a preparation of fully nonphosphorylated Vn with a preparation of fully phosphorylated Vn. For this comparison, we used nonphosphorylated wt-Vn with CKII-phosphorylated wt-Vn. In an attempt to achieve the highest stoichiometry in the phosphorylation of wt-Vn, we carried out the phosphorylation in the presence of poly-L-lysine, which has been shown to activate CKII in vitro (53). Indeed, as seen in Fig. 5 (inset), when poly-L-lysine was included in the phosphorylation reaction mixture, the incorporation of 32 P was enhanced, and it was targeted specifically to Thr 50 and Thr 57 sites, as indicated by the finding that no incorporation of 32 P occurred in the T50A/T57A mutant (Fig. 5,  inset, lane 4). The stoichiometry of phosphorylation was calculated to be 1.7 mol of phosphate per mol of Vn. Because these two CKII phosphorylation sites in Vn are in the vicinity of its RGD sequence (positions 45-47), which is well known to be involved in cell adhesion and spreading (9, 10), we first tested the functional consequences of the CKII phosphorylation on cell adhesion. As shown in Fig. 5, the adhesion of BAECs to CKII-phosphorylated wt-Vn is significantly enhanced in comparison to the nonphosphorylated wt-Vn. It should be noted, however, that although the enhanced cell adhesion with CKII phosphorylated wt-Vn was reproducible, its quantitation was erratic, presumably because the complete removal of poly-Llysine was difficult in practice, and we found that poly-L-lysine interfered with the adhesion assays. We therefore sought further support to our finding using a molecular biology approach.
It was previously shown that mutation of serines or threonines in a protein to the negatively charged amino acid Glu yields a protein analog that often mimics the activity of that protein phosphorylated at these serines or threonines (54). The obvious advantages of such a mutant are that it constitutes a homogenous population of the phosphoprotein analog and that it can be used alongside a mutant in which the same residues are replaced by a nonphosphorylatable amino acid, such as alanine, which constitutes an appropriate control of the nonphosphorylated protein. To make use of the molecular biology approach in our case, we compared the double mutants T50E/ T57E (representing the phosphorylated Vn) and the double mutant T50A/T57A (representing the fully nonphosphorylated Vn).
[ 35 S]Methionine-labeled BAECs (or NIH-3T3 cells) were used to study the effect of introducing negatively charged amino acids (Glu residues) at positions 50 and 57 of Vn on its effectiveness in cell adhesion. The cells to be studied were placed on plates of either serially diluted wt-Vn or either one of The reaction was arrested by boiling (5 min in Laemmli's sample buffer), and samples of the reaction mixtures were loaded on an SDS-polyacrylamide gel (10% acrylamide). The gels were dried and exposed to autoradiography (bottom panel). B, stoichiometry of the phosphorylation of r-Vns by CKII. Samples of Vns phosphorylated in vitro by CKII were loaded on an SDS-polyacrylamide gel (10% acrylamide). The gel was dried and exposed to autoradiography. The phosphorylated Vn bands were excised from the dried gel and counted on a scintillation counter. The calculation of the stoichiometry of phosphorylation was calculated on basis of the specific radioactivity of [␥-32 P]ATP and the amount (in mol) of Vn applied in each lane. the mutants T50E/T57E or T50A/T57A for 30 min. In this experimental setup, the number of adhered cells was adequately monitored by the residual radioactivity remaining on the plates after washings, as described under "Materials and Methods." As seen in Fig. 6A, the adhesion of BAECs plated on T50E/T57E was severalfold higher than that of the mutant T50A/T57A (see, for example, the values at the r-Vn concentration of 7.5 nM). Similarly, the adhesion of NIH-3T3 cells to the T50E/T57E mutant was higher than that obtained with T50A/T57A (see, for example, values at the r-Vn concentration of 10 nM) (Fig. 6B). This difference in adhesion could not attributed to a difference in the r-Vn bound to the plates, because the binding of these Vns to the polystyrene plates was found to be essentially identical (see Fig. 6C). It should be noted that some enhancement in adhesion was also observed with the T50A/T57A mutant (see Fig. 6, A and B), raising the possibility that the increased adhesion observed with T50E/T57E is not due exclusively to the charge inserted at the CKII phosphorylation sites. Therefore, we concluded that a comparison between T50E/T57E and T50A/T57A is probably more adequate than a comparison of T50E/T57E to wt-Vn.
In view of the above evidence suggesting an involvement of the CKII phosphorylation of Vn in the control of its cell adhesion properties, we attempted to find out whether this phosphorylation might also modulate its involvement in cell spreading. Cells were plated on top of the different r-Vns (wt-Vn and the mutants mentioned above), and the spreading of cells was monitored under a phase microscope, with persistent warming (37°C). The rate of spreading of both BAECs and NIH-3T3 cells on the T50E/T57E mutant of Vn was significantly faster than on the T50A/T57A mutant or on wt-Vn. As seen in Fig. 7, the NIH-3T3 cells that were plated on the various immobilized Vns became attached after 10 min (unattached cells were removed after 8 min by washing). However, after 20 min, only the cells plated on immobilized T50E/T57E were spread, suggesting that the CKII phosphorylation of Vn also enhances the rate of cell spreading.
Evidence Suggesting an Involvement of ␣ v ␤ 3 in the Enhanced Adhesion of BAECs to Vn T50E/T57E-Attempting to understand the mechanism through which the CKII-phosporylation enhances the ability of Vn to promote cell adhesion and spreading, we considered two possibilities: (a) that the CKII phosphorylation modulates Vn function by enhancing the affinity between Vn and its specific integrin(s) on the cell in question; or (b) that upon phosphorylation, the binding of Vn is diverted, to another specific integrin. To find out which of these possibilities is correct, we used the integrin-specific antibodies (55), searching for the antibodies that would block the cell adhesion mediated by wt-Vn and by its two double mutants, Vn T50E/ T57E and Vn T50A/T57A. The results presented in Fig. 8A indicate that the BAEC adhesion to wt-Vn, as well as to Vn FIG. 5. Effect of CKII phosphorylated wt-Vn on BAEC adhesion compared with that of the nonphosphorylated wt-Vn. Adhesion of BAECs onto CKII phosphorylated wt-Vn and onto nonphosphorylated wt-Vn. Polystyrene plates were coated with increasing amounts of CKII-phosphorylated wt-Vn (phosphorylation in the presence of ATP (E)) and with nonphosphorylated wt-Vn (which contained all of the components of the phosphorylation reaction mixture, except that ATP was substituted by AMP-PCP (Ⅺ)) (for details, see under "Materials and Methods"). [ 35 S]Met-labeled BAECs were plated on top of the different substrata for 30 min. Cell adhesion was determined by counting the residual radioactivity after washing as described under "Materials and Methods." Inset, obtaining a high stoichiometry in the phosphorylation of wt-Vn. Samples of wt-Vn or Vn T50A/T57A were phosphorylated in vitro by CKII in the absence or presence of 15 g/ml poly-L-lysine (for details see under "Materials and Methods") and were loaded on an SDS-polyacrylamide gel (10% acrylamide). The gel was dried and exposed to autoradiography.

FIG. 6. Effect of replacing Thr 50 and Thr 57 with Glu or Ala on the Vn-mediated cell adhesion of BAECs and of NIH-3T3 cells.
Polystyrene plates were coated with increasing amounts of different r-Vns. [ 35 S]Met-labeled BAECs or NIH-3T3 cells were plated on top of the different substrata for 30 min. Cell adhesion was determined by counting the residual radioactivity after washing as described under "Materials and Methods." A, adhesion of BAECs to wt-Vn, to its double mutant Vn T50E/T57E, and to its double mutant Vn T50A/T57A. B, adhesion of NIH-3T3 cells to wt-Vn, to its double mutant Vn T50E/ T57E, and to its double mutant Vn T50A/T57A. C, determination of the r-Vns content on the polystyrene plates used in panels A and B. The comparison, carried out by enzyme-linked immunosorbent assay using anti-human Vn polyclonal antibodies, was made to secure that the differences observed in panels A and B are not due merely to differences in the adsorption efficacy of the various Vns onto the plastic plates. T50E/T57E and Vn T50A/T57A, involves in these cells the ␣ v ␤ 3 integrin, because in all three cases, the adhesion was found to be significantly inhibited by the anti-␣ v ␤ 3 monoclonal antibodies. The fact that a higher concentration of antibodies was needed to achieve a similar inhibition of adhesion to Vn T50E/ T57E can be attributed to a higher affinity of Vn T50E/T57E toward the ␣ v ␤ 3 integrin. In line with this suggestion is the fact that the differences in adhesion are almost abolished when a higher concentration of anti-␣ v ␤ 3 monoclonal antibodies was used (see the antibody concentration of 50 g/ml in Fig. 8). It should be emphasized that the lack of inhibition by the anti-␣ v ␤ 5 cannot be taken as evidence to exclude the involvement of ␣ v ␤ 5 in other cells, because the FACS analysis of the BAECs used here showed that they essentially do not express ␣ v ␤ 5 (Fig. 8B).

DISCUSSION
Protein phosphorylation is well established as a key mechanism for intracellular regulation. However, in the last few years, several reports provided evidence to show that phosphorylation might also function as a regulatory device in the cell exterior (for a review, see Ref. 26). These reports included evidence that protein kinases are present outside the cell as ectoenzymes (on the cell surface) or exoenzymes (detached from the cell) and that they may well have outside the cell appropriate peptide or protein substrates (24, 25, 34 -37). In addition, the occurrence of a release of ATP (a co-substrate of the kinases) into the cell exterior (56), the physiological circumstances under which the kinases and their substrates become exposed to each other, and, finally, the fact that phosphorylation modulates the functional properties of some of the kinase substrates support the notion that extracellular phosphorylation may well be a physiological regulatory mechanism. For example, we have shown that Vn is functionally modulated by PKA, which is released from platelets upon their physiological stimulation with thrombin (24 -26), and similarly, that PKC phosphorylation of Vn attenuates its cleavage by plasmin (32).
Evidence for the existence of an extracellular CKII activity was documented in several laboratories. Such kinase activity was found, for example, on epithelial cells (34,35), on neutrophils (36,37), on platelets (40,41), and on endothelial cells (38,39,57). As in the case of PKA mentioned above, it was also shown that cells release a CKII-like activity in response to thrombin stimulation (39). Therefore, circulating blood proteins might well be physiological substrates for extracellular CKII. Indeed, the coagulation cofactors Va and VIII were shown to be substrates for the platelet-released CKII (40,41), and the phosphorylation of fibrinogen by CKII was found to have an effect on the structure of the fibrin fibers formed after the fibrinogen cleavage by thrombin (58 -60).
In this paper, we showed (i) that Vn is a substrate for CKII; (ii) that the CKII phosphorylation of Vn is selectively targeted to two Thr residues, Thr 50  Polystyrene plates were coated with r-Vns (each at 5 g/ml). [ 35 S]Metlabeled BAECs were preincubated (with shaking) for 30 min at 22°C, in the presence of increasing amounts of monoclonal antibodies against ␣ v ␤ 3 (filled symbols) and against ␣ v ␤ 5 (open symbols) before the adhesion assay was performed. The BAEC adhesion was determined by the residual radioactivity on the plates after extensive washing, as described under "Materials and Methods." The percentage of adhered cells was calculated in comparison to a control of nonrelevant anti-hemagglutinin monoclonal antibodies. f and Ⅺ, wt-Vn; OE and ‚, Vn T50A/ T57A; q and E, Vn T50E/T57E. B, FACS analysis to determine the ␣ v ␤ 3 and ␣ v ␤ 5 integrins present in BAECs. BAECs were incubated with monoclonal antibodies directed against either ␣ v ␤ 3 or ␣ v ␤ 5 , followed by incubation with the secondary antibodies, fluorescein isothiocyanateconjugated goat anti-mouse IgG. Control BAECs were incubated with the secondary antibody only, as described under "Materials and Methods." The FACS analysis was performed with a Becton Dickinson FAC-Scan, using the 530 filter. For each antibody, 5000 cells were analyzed.
Vn; (iii) that the CKII phosphorylation of Vn is biochemically feasible in vivo, as it has a K m of 0.5-2 M, lower than the Vn concentration in blood (3-6 M); (iv) that the phosphorylation of Thr 57 facilitates the phosphorylation of Thr 50 , presumably by converting Thr 50 into a more preferred site for CKII; (v) that the stoichiometry of the CKII phosphorylation of plasma-Vn reaches a plateau at ϳ0.35 mol/mol), suggesting that in vivo, plasma-Vn is partially prephosphorylated (note, however, that at this stage it is not clear whether the CKII phosphorylation occurs intra-or extracellularly); (vi) that phosphorylation of Vn at positions Thr 57 and Thr 50 results in a significant modulation of one of its key physiological functions, cell adhesion and spreading; and (vii) that in BAECs, the enhancement of cell adhesion by the Vn T50E/T57E is mainly due to an increased affinity toward the ␣ v ␤ 3 receptors.
The major implication of the findings reported here is that the CKII phosphorylation of Vn modulates a major physiological function of this protein, cell adhesion and spreading, and that this phosphorylation actually converts Vn from a cellular "glue" to a cellular "super glue." The exact mechanism involved in this modulation is not clear yet. However, we assume that the CKII phosphorylation of Vn (as in many other proteins) modulates the Vn structure in a way that favors the conformation(s) that exhibits the desired biological activity. In the case of Vn, this may be an "open" conformation or a "multimeric" form of this adhesive protein. It is commonly accepted that prior to the posttranslational modification, there is an equilibrium in which a certain (probably very low) percentage of the molecules exist in this active conformation. This percentage is usually responsible for the "basal" activity of an enzyme, for example. The role of the posttranslational modification is to establish a new equilibrium in which there is a greater percentage of molecules in the "active" conformation and consequently an increased rate of cell spreading. Further work is needed to establish that this modulation occurs in vivo as a physiological means of controlling the attachment and detachment of cells, and possibly in cell migration. We do hope that this report will set the stage for such study, which may also have important clinical implications.
Cell adhesion and spreading is now known to be involved in many diseases, including cancer (invasion and metastasis), thrombosis, inflammation, osteoporosis, proliferative retinopathy, etc. (61)(62)(63)(64)(65). In such diseases, cells often show alterations in their adhesive properties (66). Of special interest is the involvement of integrins in cancer (67). It was found that various tumor types stop producing specific integrins or display a different population of integrins, facilitating the migration of cells that normally do not migrate, providing them with means for invasion, and promoting the formation of new blood vessels (angiogenesis), thus giving the tumor access to the bloodstream and to nourishment (68 -71). Molecular analysis of the specific integrins involved in these pathological conditions, and elucidating the regulatory mechanisms responsible for the control of cell adhesion to the substratum, or to each other, might shed light from a new angle on these diseases and be useful in the development of new drugs to challenge them.