Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin þ perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM-and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express 1casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete f-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine ,3casein gene. The expression of CAT in these lines demonstrated a strking matrix and hormone dependency (>150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the (-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous (-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.Mammary epithelial cells provide a prime example for the dramatic effect of extracellular matrix (ECM) on morphology (1-3) as well as milk protein gene expression (2, 4-7). The molecular mechanisms involved in this interaction, however, are far from understood.Two approaches have been taken to address this problem: (i) To determine the nature of ECM-mammary cell interaction at the cell surface (8) and (ii) to determine the nature of cis-and trans-acting sequences and factors involved in transcriptional regulation. The absence offunctional cell lines for stable transfection of chimeric genes has been a major impediment in the latter approach. While transgenic animals have been used for studies ofhormonal regulation (9), the role of the ECM in tissue-specific gene expression cannot be easily studied in such models.With the availability of the functional COMMA-1D mammary epithelial cell strain derived from midpregnant mice (10), a number of laboratories have attempted to study the regulatory sequences of milk protein genes in response to lactogenic hormones. Some earlier attempts to transfect casein constructs failed, as the transfected cell bypassed hormonal regulation (11). In HC11 cells, a cloned derivative of COMMA-1D cells, Doppler et al. (12,13) succeeded in dissecting aspects of the hormone-responsive cis-acting elements of the -3-casein gene. These cells, however, have lost their ability to respond to substrata and theref...

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate f-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of 3-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes f-casein unidirectionally into a lumen. We have further shown that ECMand prolactin-dependent regulations of f-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine ,B-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the 3-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin-and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive f-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin-and ECMmediated regulation.

We have previously described a 160-bp enhancer (BCE-1) in the bovine ␤-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-␤ and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin-and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-␤ but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.It is now well established that the processes of development and differentiation depend on a cell's ability to correctly perceive its microenvironment (reviewed in references 1 and 43). A key component of this environment is the extracellular matrix (ECM). The ECM is an organized network of glycoproteins, proteoglycans, and glycosaminoglycans, components important for cell morphology as well as for signal transduction via cell surface integrins and ultimately for tissue-specific gene expression (reviewed in reference 43).The mammary gland appears to be particularly well suited for the study of ECM-induced differentiation and gene expression. In the adult animal, the gland develops after puberty and functionally differentiates in response to pregnancy. The mechanisms involved in these developmental processes are complex and guided by various hormones (54), growth factors (53), and the ECM (3). Milk protein expression is initiated at mid-pregnancy and correlates with the synthesis and deposition of a specialized laminin-rich ECM during alveolar development. Expression of these milk proteins can be used as markers for the differentiated state of the gland. In the last decade, a number of model systems using mammary epithelial cells to study ECM-dependent gene regulation have been developed. These range from primar...

By complementation of the cyri-) mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1). The isolated DNA restored adenylate cyclase activity to cyri-) mutants and directed integration at the CYR] locus. Wild-type strains transformed with CYR] DNA on the high copy number vector YEp24 contained 4-to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert. This result suggests that expression of the CYRI gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S. cerevisiae. CYR)-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyri-) and maps to the same locus. Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP. These observations support the view that CDC35 and CYR] are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.Adenosine 3',5'-monophosphate (cAMP) is thought to play at least two important regulatory roles in the yeast Saccharomyces cerevisiae: As a negative regulator of sporulation in a/a diploids (1) and as a positive regulator at the "start" position of the cell division cycle (2). The MATERIALS AND METHODSStrains, Plasmids, and Media. Table 1 shows the yeast strains used. Strain GC26-7B was derived from a cross between HR125-5Da and AM18-5C, followed by two backcrosses of cyrl-1 segregants to HR125-5Da or 1369. NW23-6D and NW23-9C were similarly obtained from a cross between GC26-7B and 1369 and two additional backcrosses of cyrl-J segregants to HR125-5Da or 1369. NW56-8A was obtained as an Ade-Cyr' cam segregant from a cross between AM3-4B and NW23-9C with three subsequent backcrosses of such segregants to NW23-6D or NW23-9C. NW33-1 was a ura3-52 cdc35-1 segregant from a cross between BR314-4A and 1369. The Escherichia coli strain was HB101.Plasmids used were YEp24 [pBR322 containing the yeast URA3 gene and a portion of yeast 2-,um DNA (10)] and YIp5 [pBR322 containing the yeast URA3 gene (11)].Rich medium for cultivation of yeast was YEPD (1% yeast extract/2% Bacto-peptone/2% glucose). Defined medium was 6.7 g of yeast nitrogen base without amino acids per liter and 2% glucose, supplemented with appropriate factors to support growth of auxotrophic strains. Solid medium contained 2% Bacto-agar. Media for growth of cyri-l mutants were supplemented with 2 mM cAMP.Preparation of Yeast Particulate Extracts. Strains to be tested for adenylate cyclase activity were grown in defined medium lacking uracil to maintain the URA3 plasmids. Cells (5 x 108) were harvested at a density of 1-2 x 107 per ml, washed once with d...

Purpose: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models.Experimental Design: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated.Results: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3′-[18 F]fluoro-3′-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3′-[ 18 F]fluoro-3′-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. Conclusions: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials. Clin Cancer Res; 16(21); 5177-88. ©2010 AACR.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates.Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene.The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.