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Cheryl A Conover

Publications and source records attributed to Cheryl A Conover.

33 records · Page 2Linked to original sources
PubMed2003

The insulin-like growth factor/insulin system in epithelial ovarian cancer.

Ovarian cancer is the most lethal of the gynecologic malignancies, in part because the lack of striking symptoms in many women often results in diagnosis only after the disease has spread. In order to develop rational biologic or pharmacologic therapies, it is important to understand the biology of both normal and malignant ovarian epithelial cells. This review will summarize current knowledge of the expression and function of proteins in the insulin-like growth factor system in ovarian epithelium, including the peptide ligands, receptors, binding proteins, and binding protein proteases. All components of this complex regulatory system are present in the ovarian epithelium and may play important roles in regulating the normal biology of ovarian surface epithelial cells or the transformed phenotype of ovarian carcinoma cells.

Animals↗
PubMed2003

Effects of insulin-like growth factor-I on cultured human coronary artery smooth muscle cells.

The growth-promoting effects of insulin-like growth factor-I (IGF-I) appear to be different in vascular smooth muscle cells from various segments of the arterial tree. Little information exists on human coronary artery smooth muscle cells (CoSMC), the primary elements of coronary atherosclerosis and post-angioplasty restenosis. In this study we determined the effects of IGF-I on cultured human CoSMC. Type I IGF receptors (IGF-R) were present on CoSMC as assessed by affinity cross-linking of 125I-IGF-I to monolayer cultures. IGF-I was a weak mitogen, 1.5-fold increase in [3H]thymidine incorporation, for CoSMC. However, IGF-I had a potent motility effect on CoSMC with a 314+/-12% increase in cell migration (P<0.001), comparable to that of 5% FBS. IGF-I-stimulated motility was partially inhibited by alphaIR-3, a specific IGF-R inhibitor (P<0.05). Addition of kistrin, a disintegrin, or LM609, a specific alpha(V)beta(3) integrin neutralizing antibody, abolished IGF-I-stimulated migration (P<0.001). This study indicates that IGF-I is a potent motility agent for human CoSMC via the alpha(V)beta(3) integrin receptor, but exerts little mitogenic effect. Because CoSMC migration plays a crucial role in atherosclerosis and restenosis, IGF-I blockade has the potential to limit lumen reduction.

Cell Movement↗
PubMed2003

Role of extracellular matrix in insulin-like growth factor (IGF) binding protein-2 regulation of IGF-II action in normal human osteoblasts.

Insulin-like growth factor binding protein-2 (IGFBP-2) in its native form had little affinity for extracellular matrix (ECM) derived from human or rat osteoblastic cells. However, in the presence of IGFs, IGFBP-2 binding to ECM was markedly enhanced, with IGF-II being more effective than IGF-I. IGF-II-enhanced binding of IGFBP-2 to ECM was specific for IGFBP-2 of the six known IGFBPs. In the presence of IGF-II, IGFBP-2 bound with high affinity to heparin-Sepharose, but not to type I collagen, fibronectin, or laminin. Furthermore, heparin and heparan sulfate, but not chondroitin sulfate, inhibited IGFBP-2/IGF-II binding to ECM. High salt (100 mM NaCl) inhibited, while CaCl(2) enhanced binding of IGFBP-2/IGF-II to ECM. In the presence of ECM, IGFBP-2/IGF-II was as effective as IGF-II alone in stimulating [3H]thymidine and [3H]proline incorporation and in inhibiting apoptosis in cultured human osteoblasts. On the other hand, IGFBP-2 was a potent inhibitor of IGF-II action in human breast and ovarian carcinoma cells. There was no difference between soluble and ECM-associated IGFBP-2 in affinity for IGF-I and IGF-II. These data suggest a unique mechanism for targeting an anabolic IGFBP-2/IGF-II complex in bone.

Animals↗
PubMed2003

Detecting vulnerable plaque using peripheral blood: inflammatory and cellular markers.

The concept of the vulnerable patient has arrived. Enhanced diagnostic methods will eventually permit accurately finding and treating these patients and their disease. Clinical Cardiologists now recognize that coronary atherosclerosis is two pathophysiologically distinct syndromes: stable and unstable. Stable coronary syndromes result from fixed, severe stenoses limiting blood flow and causing secondary myocardial ischemia. The unstable acute coronary syndromes are frequently catastrophic and are pathophysiologically distinct. They result from different cell subsets causing vascular inflammatory syndromes rather than gradual lumen constriction by plaque. Though pathophysiologically distinct, they may show common pathophysiology when a ruptured plaque heals and progressively becomes a critical stenosis. For the present hs-CRP measurement is the strongest correlative factor for future clinical events due to arterial inflammation: myocardial infarction, unstable angina, stroke, and peripheral vascular disease in both diseased and apparently healthy, asymptomatic patients. The CRP plasma level also is the best risk assessment in patients with either stable or unstable angina, long term after myocardial infarction, and in patients undergoing revascularization therapies. One study showed the only independent cardiovascular risk indicators using multivariate, age adjusted and traditional risk analysis were CRP and Total/HDL cholesterol ratio. If CRP, IL-6, and ICAM-1 levels are added to lipid levels, risk assessment can be improved over lipids alone. The prevalence of high-risk subjects in the general population is low, amplifying diagnostic problems for vulnerable plaque. Since no test yet has high sensitivity or specificity, diagnostic errors are high, with many false positives and negatives. Sensitivity or specificity must be increased by developing a risk marker panel, or by simultaneously finding other markers that themselves are highly sensitive and specific for vulnerable plaque.

Arteries↗
PubMed2003

Heterogeneous role of caspase-8 in fenretinide-induced apoptosis in epithelial ovarian carcinoma cell lines.

The mechanism of action of fenretinide, a synthetic retinoid currently undergoing testing as a chemopreventive and chemotherapeutic agent, is incompletely understood. In the present study, fenretinide caused apoptotic changes, including DNA fragmentation and cleavage of caspase substrates, in six low-passage ovarian cancer cell lines. However, the caspase activation pathway used by this agent varied. Transient transfection of cDNA-encoding cytokine response modifier A (CrmA), a caspase-8 inhibitor, diminished fenretinide-induced death in OV177 cells. Likewise, IETD(OMe)-fluoromethylketone (fmk) inhibited fenretinide-induced apoptosis by >80% in OV177 or OV266 cells and by approximately 50% in OV17, OV167, or OV207 cells. Further analysis demonstrated that inhibition of Fas ligand, tumor necrosis factor-alpha, or TRAIL signaling with blocking reagents did not affect fenretinide-induced apoptosis, raising the possibility that fenretinide activates caspase-8 in a death receptor-independent manner. In contrast, CrmA transfection or IETD(OMe)-fmk treatment did not inhibit fenretinide-induced apoptosis in OV202 cells. These divergent behaviors did not correlate with increased levels of procaspase-10, which is relatively resistant to CrmA and IETD(OMe)-fmk, nor with the expression of procaspase-8 and -9, apoptotic protease activating factor-1, or cellular FLICE-like inhibitory protein. Similarly, fenretinide treatment increased ceramide levels equally in cells that do (OV177) and do not (OV202) rely on caspase-8 to initiate apoptosis. These results indicate that synthetic retinoids can use caspase-8 as an initiating caspase, but they also indicate unexpected heterogeneity in caspase activation pathways among closely related cell lines.

Apoptosis↗
PubMed2003

Localization and regulation of pregnancy-associated plasma protein a expression in healing human skin.

Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with alpha-smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.

Actins↗
PubMed2003

Transforming growth factor-beta regulation of the insulin-like growth factor binding protein-4 protease system in cultured human osteoblasts.

IGFBP-4 is an inhibitor of IGF-I in bone. We show that TGF-beta regulates IGFBP-4 and enhances IGF-I-stimulated growth of cultured human bone cells through increased expression of an IGFBP-4 protease, PAPP-A. This effect of TGF-beta on IGF-I bioavailability may promote local bone formation. Insulin-like growth factor binding protein (IGFBP-4) proteolysis is implicated in the regulation of local insulin-like growth factor (IGF)-I bioavailability during bone remodeling. The IGFBP-4 protease secreted by normal adult human osteoblastic (hOB) cells in culture is a novel metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). We have recently identified an inhibitor of PAPP-A, the precursor form of major basic protein (proMBP). Very little is known about the molecular regulation of this IGFBP-4 protease system. In the present study, we determined the effect of transforming growth factor (TGF)-beta and IGF-II, the two most abundant growth factors in human bone, on PAPP-A and proMBP expression in primary cultures of hOB cells. Treatment with TGF-beta resulted in time- and dose-dependent increases in PAPP-A mRNA expression, with a maximal 12-fold increase after 24 h of stimulation with 10 ng/ml TGF-beta. Increased PAPP-A levels in hOB cell-conditioned medium paralleled PAPP-A gene expression. In addition, TGF-beta completely suppressed proMBP expression. Treatment of hOB cells with IGF-II had no effect on PAPP-A or proMBP gene expression. However, IGFBP-4 proteolysis in cell-free assay was dependent on IGF-II, and there was increased IGF-II-dependent IGFBP-4 protease activity in conditioned medium from hOB cells that were treated with TGF-beta. IGF-I stimulation of hOB cell proliferation was markedly enhanced by pretreatment with TGF-beta and [Leu27]IGF-II, and this enhancement was prevented with protease-resistant IGFBP-4. In summary, TGF-beta regulates IGFBP-4 proteolysis in hOB cells through increased expression of the protease, PAPP-A, and decreased expression of the inhibitor, proMBP. However, functional activation of the IGFBP-4 protease system is dependent on IGF-II, which acts at a post-translational level. These data support a model whereby local TGF-beta and IGF-II in the bone microenvironment coordinately amplify IGF-I bioavailability through controlled IGFBP-4 proteolysis, which may be a means to promote bone formation.

Cell Culture Techniques↗
PubMed2002

Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.

Amino Acid Sequence↗
PubMed2002

Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis.

Elevated serum levels of insulin-like growth factor binding protein-2 (IGFBP-2) and a precursor form of IGF-II are associated with marked increases in bone formation and skeletal mass in patients with hepatitis C-associated osteosclerosis. In vitro studies indicate that IGF-II in complex with IGFBP-2 has high affinity for bone matrix and is able to stimulate osteoblast proliferation. The purpose of this study was to determine the ability of the IGF-II/IGFBP-2 complex to increase bone mass in vivo. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats. At the time of surgery, 14-day osmotic minipumps containing vehicle or 2 microg IGF-II+9 microg IGFBP-2/100g body weight/day were implanted subcutaneously in the neck. Bone mineral density (BMD) measurements were taken the day of surgery and 14 days later using a PIXImus small animal densitometer. Neurectomy of the right hindlimb resulted in a 9% decrease in right femur BMD (P<0.05 vs. baseline). This loss in BMD was completely prevented by treatment with IGF-II/IGFBP-2. On the control limb, there was no loss of BMD over the 14 days and IGF-II/IGFBP-2 treatment resulted in a 9% increase in left femur BMD (P<0.05). Bone histomorphometry indicated increases in endocortical and cancellous bone formation rates and in trabecular thickness. These results demonstrate that short-term administration of the IGF-II/IGFBP-2 complex can prevent loss of BMD associated with disuse osteoporosis and stimulate bone formation in adult rats. Furthermore, they provide proof of concept for a novel anabolic approach to increasing bone mass in humans with osteoporosis.

Animals↗
PubMed2002

Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity.

Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human.

Amino Acid Sequence↗
PubMed2002

Functional insulin receptors on human epithelial ovarian carcinoma cells: implications for IGF-II mitogenic signaling.

The insulin receptor mediates a proliferative response in certain transformed cells, but little is known about its function in ovarian cancer. We used human epithelial ovarian carcinoma cell lines and lifespan-extended normal ovarian surface epithelial (OSE) cells to examine (125)I-insulin binding and mitogenic responses to insulin. All cancer cell and OSE cultures specifically bound (125)I-insulin. Except for OV202, the carcinoma lines had elevated insulin binding compared with OSE cells. All carcinoma lines except OV202 expressed insulin receptor as detected by flow cytometry and increased (3)H-thymidine incorporation or cell number in response to 0.1-10 nM insulin. Interestingly, similar concentrations of IGF-II also induced proliferation of the insulin-responsive cancer cell lines and displaced (125)I-insulin binding. Direct binding of (125)I-IGF-II to the insulin receptor was visualized by cross-linking and immunoprecipitation. Binding of IGF-II to the insulin receptor and a proliferative effect of insulin suggest the presence of insulin receptor isoform A. Real-time PCR analyses confirm that insulin receptor isoform A expression predominates over isoform B expression in the ovarian carcinoma cell lines. This report suggests that the insulin receptor may play a role in the regulation of ovarian cancer cell growth.

Alternative Splicing↗
PubMed2002

Molecular regulation of the IGF-binding protein-4 protease system in human fibroblasts: identification of a novel inducible inhibitor.

The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A's proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, beta-phorbol 12,13-didecanoate (beta-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of beta-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with beta-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with beta-PDD induced pro-MBP mRNA and protein expression within 6 h. beta-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of beta-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

Blood Proteins↗
PubMed2002

Use of site-specific antibodies to characterize the circulating form of big insulin-like growth factor II in patients with hepatitis C-associated osteosclerosis.

Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome of adult-onset osteosclerosis. An understanding of the factor(s) leading to the stimulation of bone formation in these patients may provide novel anabolic approaches for the treatment of osteoporosis. We have demonstrated that HCAO patients have a specific increase in circulating big IGF-II (IGF-IIE) and IGF-binding protein-2 (IGFBP-2) levels, and that IGF-IIE and IGFBP-2 circulate together in a bioavailable, 50-kDa complex. Patients with nonislet cell tumor hypoglycemia (NICTH) also have increased circulating IGF-IIE and IGFBP-2 levels. However, HCAO patients do not exhibit hypoglycemia, nor do NICTH patients exhibit obvious osteosclerosis. Thus, to better understand the reason(s) for the differing clinical manifestations of the IGF-IIE excess in the two syndromes, we characterized IGF-IIE in HCAO and NICTH sera using recently developed antibodies (Ab) recognizing either the full-length IGF-IIE 89-amino acid C-terminal extension peptide (IIE(138-156) Ab) or specific cleavage forms of IGF-IIE (IIE(78-88) Ab and IIE(89-101) Ab). The predominant IGF-IIE form in HCAO serum migrated on SDS-PAGE as a single band at approximately 18 kDa that reacted with the IIE(89-101) Ab. On the other hand, the predominant form in NICTH serum migrated as a doublet of 14 and 16 kDa that reacted with the IIE(78-88) Ab. There results are consistent with differential processing of the IGF-IIE precursor at predicted cleavage sites producing IGF-IIE(1-104) and IGF-IIE(1-88) in HCAO and NICTH, respectively. As these two forms may have differing biological activities and/or targeting properties, our findings may explain at least in part the different manifestations of IGF-IIE overproduction in the two syndromes.

Antibody Specificity↗