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Background: Ovarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer., Methods: We aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples., Results: Our in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis., Conclusions: Our preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective., (© 2024. The Author(s).)

The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

The expression of Sialyl-Tn (STn) in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. STn is expressed on ovarian cancer biomarkers including CA-125 (MUC16) and MUC1, and elevated serum levels of STn in ovarian cancer patients correlate with lower five-year survival rates. In the current study, we humanized novel anti-STn antibodies and demonstrated the retention of nanomolar (nM) target affinity while maintaining STn antigen selectivity. STn antibodies conjugated to Monomethyl Auristatin E (MMAE-ADCs) demonstrated in vitro cytotoxicity specific to STn-expressing ovarian cancer cell lines and tumor growth inhibition in vivo with both ovarian cancer cell line- and patient-derived xenograft models. We further validated the clinical potential of these STn-ADCs through tissue cross-reactivity and cynomolgus monkey toxicity studies. No membrane staining for STn was present in any organs of human or cynomolgus monkey origin, and the toxicity profile was favorable and only revealed MMAE-class associated events with none being attributed to the targeting of STn. The up-regulation of STn in ovarian carcinoma in combination with high affinity and STn-specific selectivity of the mAbs presented herein warrant further investigation for anti-STn antibody-drug conjugates in the clinical setting., Competing Interests: This research may lead to the development of products that are owned by and/or licensed to Siamab Therapeutics, Inc. which employs DAE, JMP, RIN, JNS, DTD, and JB; and which has compensated AL as a consultant. JB owns stock in Siamab Therapeutics, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Metastatic castration resistant prostate cancer is one of the main causes of male cancer associated deaths worldwide. Development of resistance is inevitable in patients treated with anti-androgen therapies. This highlights a need for novel therapeutic strategies that would be aimed upstream of the androgen receptor (AR). Here we report that the novel small molecule anti-androgen, galeterone targets USP12 and USP46, two highly homologous deubiquitinating enzymes that control the AR-AKT-MDM2-P53 signalling pathway. Consequently, galeterone is effective in multiple models of prostate cancer including both castrate resistant and AR-negative prostate cancer. However, we have observed that USP12 and USP46 selectively regulate full length AR protein but not the AR variants. This is the first report of deubiquitinating enzyme targeting as a strategy in prostate cancer treatment which we show to be effective in multiple, currently incurable models of this disease., Competing Interests: CONFLICTS OF INTEREST None.

Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn + cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo , depleting STn + tumor cells. In summary, STn + cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn + CSC and STn + non-CSC populations., Competing Interests: CONFLICTS OF INTEREST Rosemary Foster, Silvia Fatima Hernandez, Linah Al-Alem, Chiara Bellio, Bianca Zarrella, Whitfield B. Growdon, and Kristen Starbuck have no conflicts of interest. Bo Rueda receives stock options for serving as a member of the scientific advisory committee for Siamab Therapeutics, Inc. David Eavarone, Jillian Prendergast, Jenna Stein, Daniel Dransfield and Jeffery Behrens are all employed by Siamab Therapeutics, Inc. This research may lead to the development of products which may be owned by and/or licensed to Siamab Therapeutics, Inc. in which they have a business and/or financial interest.

Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h-a much more feasible timeframe compared to previous recommendations.

Objective: In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1-MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen-induced arthritis (CIA)., Methods: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. From the onset of clinical arthritis, mice were treated with MT1-MMP selective inhibitory antibody DX-2400 and/or TNFR-Fc fusion protein. Disease progression was monitored daily, and serum, lymph nodes, and affected paws were collected at the end of the study for cytokine and histologic analyses. For in vitro analysis, bone marrow-derived macrophages were stimulated with lipopolysaccharide for 24 hours in the presence of DX-2400 and/or TNFR-Fc to analyze cytokine production and phenotype., Results: DX-2400 treatment significantly reduced cartilage degradation and disease progression in mice with CIA. Importantly, when combined with TNF blockade, DX-2400 acted synergistically, inducing long-term benefit. DX-2400 also inhibited the up-regulation of interleukin-12 (IL-12)/IL-23 p40 via polarization toward an M2 phenotype in bone marrow-derived macrophages. Increased production of IL-17 induced by anti-TNF, which correlated with an incomplete response to anti-TNF, was abrogated by combined treatment with DX-2400 in CIA., Conclusion: Targeting MT1-MMP provides a potential strategy for joint protection, and its combination with TNF blockade may be particularly beneficial in RA patients with an inadequate response to anti-TNF therapy., (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Background: Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects., Methods: Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided., Results: DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors., Conclusion: MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

MMP intervention strategies have met with limited clinical success due to severe toxicities. In particular, treatment with broad-spectrum MMP-inhibitors (MMPIs) caused musculoskeletal pain and inflammation. Selectivity may be essential for realizing the clinical potential of MMPIs. Here we review discoveries pinpointing membrane-bound MMPs as mediators of mechanisms underlying cancer and inflammation and as possible therapeutic targets for prevention/treatment of these diseases. We discuss strategies to target these therapeutic proteases using highly selective inhibitory agents (i.e., human blocking antibodies) against individual membrane-bound MMPs.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.

Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab-IGF-II complexes (M64-F02-IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 A. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 107, c = 111.5 A, and also diffracted X-rays to 2.2 A resolution.

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

LFA-1 (alpha(L)beta(2)) mediates cell-cell and cell-extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding alpha(L) I domain from low-affinity (LA), closed form, to the high-affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA alpha(L) I domain have not been described. Here, we report the identification and characterization of a human antibody AL-57, which binds to the alpha(L) I domain in a HA but not LA conformation. AL-57 was discovered by selection from a human Fab-displaying library using a locked-open HA I domain as target. AL-57 Fab-phage bound HA I domain-expressing K562 cells (HA cells) in a Mg(2+)-dependent manner. AL-57 IgG also bound HA cells and PBMCs, activated by Mg(2+)/EGTA, PMA, or DTT. The binding profile of AL-57 IgG on PBMCs was the same as that of ICAM-1, the main ligand of LFA-1. In contrast, an anti-alpha(L) murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL-57 IgG blocked ICAM-1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM-1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA-induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA-1-mediated, adhesive functions. AL-57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.

In some cell systems, the antiproliferative effects of 8-Cl-cAMP, a site-selective cAMP analog specific for the type I cAMP-dependent protein kinase, are mediated by its metabolite, 8-Cl-adenosine. These effects were once thought to be specific to transformed cells. We investigated the ability of 8-Cl-adenosine to regulate growth and differentiation in primary cultures of mouse epidermal keratinocytes. A 24 h exposure of keratinocytes to 8-Cl-adenosine inhibited [3H]thymidine incorporation in a dose-dependent manner with an apparent IC(50) of 7.5 microM, and these effects were completely reversible. To determine the ability of 8-Cl-adenosine to induce differentiation of primary keratinocytes, we measured keratin-1 expression and transglutaminase activity, markers of early and later stages of keratinocyte differentiation, respectively. Interestingly, exposure of keratinocytes to 25 microM 8-Cl-adenosine for 24 h had no effect on keratin-1 expression or transglutaminase activity. The 8-Cl-adenosine-induced growth arrest of keratinocytes required uptake of the compound and was accompanied by an increase in protein expression of the cyclin-dependent protein kinase inhibitor p21(WAF1/Cip1). These results demonstrate that 8-Cl-adenosine inhibits growth in a non-transformed/non-immortalized cell system, possibly through an elevation in p21(WAF1/Cip1) protein levels, without inducing differentiation.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.

8-Cl-adenosine represents a novel nontoxic chemotherapeutic agent shown to inhibit growth of a number of colorectal cancer cell lines. We have utilized the mucin-secreting colorectal cancer cell line, LS174T, to assess the growth inhibitory properties of 8-Cl-adenosine independent of its parental compound, 8-Cl-cAMP. Conversion of 8-Cl-cAMP to 8-Cl-adenosine is required for growth inhibition in LS174T cells. 8-Cl-Adenosine inhibited growth by inducing a G1 cell cycle arrest that was associated with large (eightfold) increases in p21WAF1/Cip1 and p53 protein levels and a decrease in the phosphorylation status of the retinoblastoma protein. LS174T cells did not undergo apoptosis. In addition, 8-Cl-adenosine also induced some degree of enterocytic differentiation. Both villin protein levels as well as alkaline phosphatase activity rose (2- and 3.5-fold, respectively) in response to treatment with 8-Cl-adenosine. Our results suggest that in LS174T cells, 8-Cl-adenosine not only serves as a growth inhibitory agent but also as an inducer of enterocytic differentiation.

The purine ring system is undoubtedly one of the most ubiquitous heterocyclic ring systems in nature as it has the distinction of being the parent ring in countless derivatives of biological relevance. It is not surprising then that modified purines possess the potential to impact several areas, including a better understanding of the biological effects of DNA damaging agents, enzyme/substrate interactions, and in the development of more potent medicinal agents. One focus for our research at Georgia Tech has centered around the design and synthesis of a series of extended purine analogues containing a heterocyclic spacer ring, with sites set on investigations into their use as (i) potential anticancer and antiviral agents, (ii) dimensional probes for enzyme and coenzyme binding sites, and (iii) structural probes of the minor groove of DNA. The synthesis and preliminary antitumor activity of two thieno-separated purine analogues are described herein. Tricyclic 1 was synthesized in 12 steps from tribromoimidazole and with an overall yield of 7%. Tricyclic 2 was synthesized in 9 steps with an overall yield of 13%. Both 1 and 2 exhibited growth inhibitory effects on HCT116 colorectal cancer cells in vitro.

The cAMP analogue 8-Cl-cAMP induces apoptosis and inhibits proliferation of a wide variety of malignancies in vitro and in vivo with relatively little toxicity. The antitumor effects of this compound are thought to involve its ability to modulate type I protein kinase A (PKAI). However, a nontoxic metabolite of 8-Cl-cAMP, 8-Cl-adenosine, with no known activity against PKAI, exerts growth inhibitory effects in breast, ovary, pancreas, and colorectal cancer cells in vitro and accumulates in xenografted tumors after 8-Cl-cAMP treatment in vivo. To characterize further the antitumor effects of 8-Cl-adenosine in colorectal cancer, we examined its effects on cell growth in vitro (cell number, 3H-thymidine incorporation, and soft agar colony formation) using the isogenically matched colorectal cancer cell lines HCT116, HCT116-E6 (p53-depleted), and 80S14 (p21WAF1/Cip1-null). 8-Cladenosine inhibited cell growth by 89%, 74%, and 79%, respectively in HCT116, HCT116-E6, and 80S14 cells after a 72-hour exposure. Growth inhibition coincided with DNA endoreduplication and subsequent apoptosis. Furthermore, nontoxic doses of 8-Cl-adenosine administered i.p. twice weekly for 4 weeks to athymic mice suppressed growth of HCT116-derived xenografts by 50%. These results show that 8-Cl-adenosine exerts antitumor activity against colorectal cancer independent of p53 and p21WAF1/Cip1.

Glucose-dependent insulinotropic peptide (GIP) potentiates glucose-induced insulin secretion. In addition, GIP has vasoconstrictive or vasodilatory properties depending on the vascular bed affected. In order to assess whether this effect could be related to differences in GIP receptor expression, several different endothelial cell types were examined for GIP receptor expression. GIP receptor splice variants were detected and varied depending on the endothelial cell type. Furthermore, stimulation of these cells with GIP led to cell type dependent differences in activation of the calcium and cAMP signaling pathways. To our knowledge this is the first physiological characterization of receptors for GIP in endothelial cells.

The protein kinase A (PKA) is classified as type I or II depending on the association of the catalytic subunit with either the R(I) or R(II) regulatory subunits. Alterations in the levels of these regulatory subunits and PKA activity itself appear to affect cellular proliferation and tumorigenesis. We examined colorectal tumor specimens from 45 patients to investigate the potential role of cAMP-related signaling molecules in regulating tumorigenesis. Western blot analysis (PKA subunit protein levels) and in vitro kemptide phosphorylation assays (PKA catalytic subunit activity) were performed on human colorectal tumor tissue homogenates. R(I)beta protein levels were decreased 200% in ascending and 50% in descending colonic tumors compared to adjacent mucosa. R(II) protein levels were decreased 77% in descending colonic tumors but no change was observed in ascending colonic tumors compared to adjacent mucosa. PKA activity and the absolute amount of catalytic subunit protein in ascending and descending tumors were unchanged compared to adjacent mucosa. Differences in cAMP-related signaling molecules exist between neoplastic and normal colorectal tissues. These differences may not only serve as potential therapeutic targets for chemotherapeutic agents, but also lead to the identification of novel regulatory mechanisms involved in cellular proliferation and tumorigenesis.

Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. We have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants were also identified. Both of the major splice variants showed tissue- and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that AKAP350 was associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not alter its localization. During the cell cycle, AKAP350 was associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrated noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types.

Alterations in intracellular oxidative status activate several signal transduction pathways resulting in distinct patterns of gene expression. Treatment of colorectal cancer cells with antioxidants can lead to apoptosis by induction of p21 through a mechanism involving CCAAT/enhancer-binding protein beta (C/EBPbeta). Herein, we demonstrate that the antioxidant pyrrolidinedithiocarbamate activates cAMP-dependent protein kinase (PKA) in a colorectal cancer cell line DKO-1. Activation of PKA phosphorylates Ser299 within C/EBPbeta, which is essential for protein translocation to the nucleus. Pharmacological inhibition of PKA and mutation of Ser299 to alanine blocks C/EBPbeta nuclear translocation and induction of p21. Our results indicate that a cAMP-dependent phosphorylation of C/EBPbeta at Ser299 is critical for nuclear translocation of this protein and its subsequent transactivation of genes in response to antioxidant treatment.

The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single RII-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the RII-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical RII-binding region between amino acids 503 and 516. Incubation of [32P]RII with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [32P]RII to the recombinant AKAP120 fragments that demonstrated RII binding. In vitro RII-binding affinity studies indicated a high-affinity interaction between AKAP120 and RII with a Kapp between 50 and 120 nM for the three recombinant fragments that bound [32P]RII. RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate RII subcellular sequestration in this cell type.

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.

In the glomerular mesangial cell, platelet-derived growth factor (PDGF) activates several signal transduction pathways. We examined the effect of PDGF on cAMP production and on cAMP-dependent protein kinase (A-kinase) activation. In mesangial cells, PDGF stimulated cAMP production in a dose- and time-dependent manner. This effect of PDGF was not prevented by pre-incubation with 50 microM indomethicin. PDGF also activated type I A-kinase, the predominate A-kinase isoform in mesangial cells, measured either by a decrease in A-kinase photoaffinity labeling with 8-azido-[32P]-cAMP, or by an increase in A-kinase substrate phosphorylation. The activation of A-kinase by PDGF is not dependent on the intermediate production of prostaglandins or cGMP. These data suggest that A-kinase participates in PDGF-induced signaling events in mesangial cells.

Recent investigations have suggested that subcellular compartmentalization of second messenger responsive enzyme systems may be responsible for specific patterns of cellular activation. The type II cAMP-dependent kinase (A-kinase) is localized to particular subcellular domains through the binding of the regulatory subunit (RII) dimer to A-kinase anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have investigated the presence of AKAPs throughout the gastrointestinal tract, with specific emphasis focused on the gastric parietal cell. All gastrointestinal tissues contained at least one detectable AKAP (60 kDa), with five AKAPs (50-140 kDa) in fundic and antral mucosa. Isolated gastric glands contained four AKAPs. Two AKAPs (50 and 78 kDa) were detected in purified parietal cells, with the 78 kDa AKAP (AKAP78) specific to parietal cell enriched populations. RII-binding to all of these AKAPs was abolished by preincubation of [32P]RII with a synthetic peptide representing the RII-binding region of the AKAP, HT-31. AKAP78 was distributed throughout all membrane fractions of subfractionated parietal cells, with the largest amount of RII-binding detected in the light membrane fraction. Identification of A-kinase regulatory subunits by photoaffinity labeling with 8-azido-[32P]cAMP demonstrated that RII segregated into the same parietal cell subfractions as AKAP78. A majority (approximately 60%) of AKAP78 was detected in the Triton X-100-insoluble fraction, suggesting that this protein resides in a cytoskeletal domain. AKAP78 may be involved in localizing the type II A-kinase to specific intracellular locations in the parietal cell.

Activation of either the adenylate cyclase pathway with forskolin or the guanylate cyclase pathway with sodium nitroprusside fails to induce active relaxation of serotonin-precontracted human umbilical artery smooth muscle (HUASM) but causes active relaxation of serotonin-precontracted bovine carotid artery smooth muscle (BCASM). This difference in response appears to be unique to HUASM in that all other vascular muscles exhibit relaxation in response to these substances. Forskolin and sodium nitroprusside stimulation leads to respective increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations in HUASM and BCASM. cAMP- and cGMP-dependent protein kinases are both present in HUASM and can be activated in homogenates of HUASM by the addition of exogenous cAMP and cGMP, respectively. When either forskolin or nitroprusside acts in BCASM, two low-molecular-weight proteins display an increase in the extent of phosphorylation. Neither protein shows such an increase when HUASM is treated with either agent. Thus the inability of HUASM to display active relaxation appears to be secondary to impaired activation of cyclic nucleotide-dependent protein kinases. The refractoriness to active relaxation may contribute to the vasospasm that occurs in the umbilical vasculature with parturition.

Isolated hepatocytes incubated under conditions of "chemical hypoxia" (KCN + iodoacetic acid) exhibited a marked dephosphorylation of the cytoplasmic and mitochondrial adenine nucleotides to AMP. Cytoplasmic adenine nucleotide levels (ATP + ADP + AMP) were decreased by 40%. There was no significant change in the mitochondrial adenine nucleotide pool size. For starved rats, but not for fed rats, addition of KCN to isolated hepatocytes resulted in a shift of the mitochondrial adenine nucleotide species to AMP. This difference was correlated with the maintenance of a substantial level of cytoplasmic ATP in the fed vs starved condition. The addition of fructose (but not glucose) to hepatocytes isolated from a starved rat, prevented the KCN-induced dephosphorylation of mitochondrial adenine nucleotides to AMP. Fructose-treated cells had a significant level of ATP in the cytoplasm, whereas glucose-treated cells did not. Addition of A23187 to fructose-treated (but not glucose-treated) cells resulted in a net loss in the mitochondrial adenine nucleotide content. The results suggest that the shift of matrix adenine nucleotides from ATP and ADP to AMP preserves the mitochondrial adenine nucleotide pool size during transient hypoxia by preventing net adenine nucleotide transport to the cytoplasm via the ATP-Mg/Pi carrier. This effectively protects those adenine nucleotides from the cytoplasmic purine degradation pathway, a strategy that has the potential to facilitate rapid recovery of bioenergetic status by oxidative phosphorylation upon reoxygenation.

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.