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H K Kleinman

Publications and source records attributed to H K Kleinman.

At least 91 records · Page 5Linked to original sources
PubMed1996

Laminin stimulates expression of two mitochondrial proteins during neurite outgrowth.

Differential hybridization was utilized with mRNA from NG108-15 cells cultured on either tissue culture plastic or laminin for 4 hr to identify genes whose mRNA was increased by laminin, a potent stimulator of neurite outgrowth. Two of the 16 laminin-induced clones, cytochrome b and chargerin II, are mitochondrial proteins. Northern blotting confirmed that laminin increased the mRNA levels of cytochrome b and chargerin II several fold. Antibody to chargerin II stained both processes and cell bodies of the cerebellar Purkinje cells and localized in the mitochondria of NG108-15 cells, which also showed increased protein levels in the presence of laminin. In addition, higher levels of chargerin II protein were detected in the newborn brain compared to the adult. However, inhibitors of mitochondrial protein synthesis did not affect laminin-mediated neurite outgrowth. These data suggest that the increased synthesis of mitochondrial enzymes observed with laminin treatment may not be necessary for the formation of neurites.

Animals↗
PubMed1996

The laminins.

The laminins are a family of extracellular matrix glycoproteins localized in the basement membrane that separates epithelial cells from the underlying stroma. They are also found in basement membrane surrounding fat, muscle and peripheral nerve cells. The laminins are large trimeric glycoproteins comprising three disulphide-bonded chains. They are the first extracellular matrix molecules to be observed in the developing embryo and have potent biological activities. In addition, there have been new developments in the number and localization of the homologues of the laminin chains and the role of laminin in neuromuscular disease. Their primary role is in cell-matrix attachment, but many additional biological activities, including promoting cell growth and migration, tumour growth and metastases, neurite outgrowth, nerve regeneration, wound repair and graft survival, have been demonstrated. Many of these biological activities are duplicated by proteolytic fragments of laminin and by small laminin-derived synthetic peptides. These laminin-derived peptides may be useful clinical reagents for accelerating wound healing with minimal scarring or for blocking tumour metastases.

Animals↗
PubMed1996

Overexpression of the 67 kD laminin receptor correlates with the progression of gastric carcinoma.

This retrospective study was designed to investigate the relationship between overexpression of the 67 kD laminin receptor (67LR) using immunohistochemistry, and several clinicopathological parameters including overall survival in human gastric adenocarcinoma. We stained paraffin-embedded sections of 93 resected primary gastric adenocarcinomas using a polyclonal antibody specific for the 67LR as well as monoclonal antibodies for p53 protein, epidermal growth factor receptor, proliferating cell nuclear antigen, carcinoembryonic antigen and chromagranin. The results showed statistically significant correlations between overexpression of the 67LR and types of early or advanced gastric carcinoma (p < 0.001), depth of invasion (p < 0.001), WHO histopathologic classification (p < 0.001), stage (p = 0.001), expression of p53 protein (p = 0.019), expression of epidermal growth factor receptor (p < 0.001) and proliferating cell nuclear antigen labeling index (p = 0.002). A lower proportion of signet ring cells revealed a higher percentage of overexpression of 67LR in both early (p < 0.002) and advanced (p < 0.001) gastric carcinomas. Intestinal type adenocarcinoma (according to Lauren's classification) revealed a higher percentage of overexpression of the 67LR than the diffuse type in both early (p = 0.057) and advanced (p < 0.001) gastric carcinomas. The correlations between overexpression of the 67LR and lymph node metastasis were statistically significant (p < 0.07). Although the overexpression of the 67LR tended to correlate with lower survival rates, the correlation was not statistically significant due to the limited sample size. Our data revealed that overexpression of the 67LR is correlated with the progression of gastric carcinoma. The expression of the 67LR may be important as one of the steps which determines invasiveness during the progression of cancer.

Adenocarcinoma↗
PubMed1996

Prostate specific antigen and androgen receptor induction and characterization of an immortalized adult human prostatic epithelial cell line.

Progress in prostate cancer research has been hindered by the lack of well characterized, immortalized, human prostatic epithelial cell lines that express markers of normal prostatic epithelial cells and mimic normal growth and differentiation responses to androgens. The objectives of this study were to: (i) establish immortalized cell lines from non-neoplastic, adult human prostatic epithelium using adenovirus-12/simian virus-40 (Ad12-SV40) hybrid virus; (ii) establish their prostatic epithelial origin; (iii) demonstrate androgen responsiveness; and (iv) examine response to growth factors. Primary epithelial cell cultures derived from a non-neoplastic, adult human prostate were infected with the Ad12-SV40 virus. Several immortalized clones were isolated. Single cell cloning of one clone, free of cytopathic effects, gave rise to the PWr-1E cell line. An immortalized cell line PWR-1E, which expresses many characteristics of normal prostatic epithelial cells was established. Immunostaining showed that cells express cytokeratins 8 and 18 normally expressed by differentiated, secretory prostatic epithelial cells. The most remarkable characteristics of PWR-1E cells are growth stimulation, increased expression of androgen receptor and induction of prostate specific antigen (PSA) expression in response to androgens, which indisputably establish their prostatic epithelial origin. They are positive for SV40 large-T antigen and show strong nuclear staining for p53. Cells from passages 23 and 40 were not tumorigenic in nude mice even when co-injected with Matrigel. They grow in a serum-free defined medium and respond to EGF, bFGF and TGF-beta. Passage 42-cells showed a human male (XY), hyperdiploid karyotype. The PWR-1E cell line is the only known Ad12-SV40-immortalized human prostatic epithelial cell line. PWR-1E cells can be used to study (i) the etiology and the multistep process of carcinogenesis and tumor progression in the human prostate; (ii) normal prostate physiology and differentiation; and (iii) potential prostate cancer chemopreventive agents.

Androgens↗
PubMed1996

Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells.

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.

Cell Culture Techniques↗
PubMed1996

Role of laminin-1 and TGF-beta 3 in acinar differentiation of a human submandibular gland cell line (HSG).

Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P < or = 0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-beta 3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary gland-specific protein expression in vitro.

Apoptosis↗
PubMed1995

A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1.

The basement membrane protein laminin and the IKVAV-containing sequence from the laminin alpha 1 chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110-kd IKVAV-binding protein (LBP110) isolated from brain appears to be a member of the beta-amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150-10153, 1993). In the preparation of this binding protein, a contaminating IKVAV-binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/ApP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell-surface nucleolin, an IKVAV-binding protein, suggests that this protein may function in signalling by extra-cellular matrix.

Amino Acid Sequence↗
PubMed1995

Neurotoxicity of the human immunodeficiency virus type 1 tat transactivator to PC12 cells requires the Tat amino acid 49-58 basic domain.

The acquired immunodeficiency syndrome (AIDS) frequently involves the central nervous system (CNS) and manifests as dementia due to encephalitis or diffuse neurodegeneration. Human immunodeficiency virus type 1 (HIV-1) proteins, potentially transported into the CNS by mononuclear inflammatory cells, have been implicated in the etiology of this HIV-1 associated neurological dysfunction. Here we investigate the neurotoxicity of the essential HIV-1 regulator protein Tat in vivo after microinfusion into the rat brain and in vitro using PC12, NG108-15, and GT17 neuronal cell lines. Infusion of either chemically synthesized Tat (Tat86) or recombinant Tat (rTat) into the striatal gray matter in Sprague-Dawley rats resulted in postural deviation ipsilateral to the infusion, a clinical presentation in rats associated with complete striatal dysfunction. Histologic examination 3 days after infusion revealed massive necrosis in the area of the distribution of the infusion. Infusion of heat denatured rTat, peptide Tat49-58, or peptide Tat57-86 did not result in clinically or histologically detectable brain damage. After 3 days incubation in vitro, the lethal dose for half (LD50) of PC12 cells due to rTat was 5 micrograms/ml. The LD50 for Tat86 under the same conditions was 10 micrograms/ml. Tat49-58 and Tat57-86 peptides were not toxic in vitro even at 10-fold higher doses. At 5 micrograms/ml, rTat was toxic to 100% of GT17 cells after 24 hr. At 5 micrograms/ml, Tat86 was toxic to 90% of the NG108-15 cells after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1995

Identification of cell binding sites in the laminin alpha 1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides.

The laminin alpha 1 chain carboxyl-terminal globular domain has been identified as a site of multiple biological activities. Using a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads that covered this domain, we found 19 potential active sequences. Corresponding synthetic peptides were evaluated for direct cell attachment, spreading, and inhibition of cell spreading to a laminin-1 substrate using several cell lines. Five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. Cell spreading on AG-10 was inhibited by beta 1 and alpha 6 integrin antibodies and on AG-32 was inhibited by beta 1, alpha 2, and alpha 6 integrin antibodies. In contrast, cell adhesion and spreading on peptide AG-73 were not inhibited by these antibodies. The minimum active sequences of AG-10, AG-32, and AG-73 were determined to be SIYITRF, IAFQRN, and LQVQLSIR, respectively. These sequences are highly conserved among the different species and different laminin alpha chains, suggesting that they play a critical role for biological function and for interaction with cell surface receptors.

Amino Acid Sequence↗
PubMed1995

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

Amino Acid Sequence↗
PubMed1995

Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo.

Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor-induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis.

Amino Acid Sequence↗
PubMed1995

The laminin alpha 1 chain Ile-Lys-Val-Ala-Val (IKVAV)-containing peptide promotes liver colonization by human colon cancer cells.

Laminin, a major basement membrane-specific glycoprotein, promotes the attachment, migration, and invasion of a variety of tumor cells. Since laminin is present in the perisinusoidal matrix of the liver, we studied its effects on liver colonization by human colon cancer cells (HM7, LiM6) previously shown to have liver-metastasizing ability in athymic mice. These malignant cells expressed high levels of a 32-kDa laminin-binding protein on Western blot analysis when compared to the low metastatic parental cell line. Coinjection of laminin alpha chain-derived peptides which contain the amino acid sequence Ile-Lys-Val-Ala-Val (IKVAV) significantly stimulated liver colonization as determined by liver weight (P < 0.005) and number of tumor nodules (P < 0.02) 3 weeks after splenic-portal inoculation into nude mice. No stimulation was seen with a control peptide containing the same amino acids but in a scrambled sequence. In contrast, the Tyr-Ile-Gly-Ser-Arg peptide from the laminin beta 1 chain significantly inhibited HM7 liver colonization. These differences were not due to alterations in the number of cells initially reaching the liver as determined by injection of [125I]iododeoxyuridine-labeled tumor cells, but retention in the liver was stimulated by the IKVAV-containing peptides. Flow analysis indicated that the IKVAV peptide may act, in part, by stimulating homotypic adhesion of tumor cells. These data suggest that interactions of colon cancer cells with the IKVAV site on laminin may play a role in the formation of metastatic foci in the liver through cell-cell or cell-substratum interactions which promote metastasis.

Adenocarcinoma↗
PubMed1995

Structure-activity study of a laminin alpha 1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV).

The IKVAV sequence, one of the most potent active sites of laminin-1, has been shown to promote cell adhesion, neurite outgrowth, and tumor growth. Here we have determined the structural requirements of the IKVAV sequence for cell attachment and neurite outgrowth using various 12-mer synthetic peptide analogs. All-L- and all-D-IKVAV peptides showed cell attachment and neurite outgrowth activities. In contrast, all-L- and all-D-reverse-sequence peptides were not active. Some of the analogs, in which the lysine and isoleucine residues of the IKVAV peptide were substituted with different amino acids, promoted cell attachment, but none of the analog peptides showed neurite outgrowth activity comparable to that of the IKVAV peptide. These results suggest that the lysine and isoleucine residues are critical for the biological functions of the IKVAV peptide.

Amino Acid Sequence↗
PubMed1995

Laminin SIKVAV peptide induction of monocyte/macrophage prostaglandin E2 and matrix metalloproteinases.

The laminin-derived synthetic peptide containing the SIKVAV (Ser-Ile-Lys-Val-Ala-Val) amino acid sequence has been previously shown to regulate tumor invasion, metastasis, and angiogenesis. Here, we demonstrate that this peptide also modulates human monocyte responses. Moreover, the monocytic responses elicited by this peptide are influenced by the culture conditions. When elutriated monocytes were cultured on SIKVAV substrate or in suspension with this peptide, the synthesis of prostaglandin E2, interstitial collagenase, and gelatinase B was induced and was further enhanced in the presence of concanavalin A (ConA). However, when monocytes were adhered before adding soluble SIKVAV, the peptide alone failed to induce the production of prostaglandin E2 or matrix metalloproteinases. If adherent monocytes were exposed to SIKVAV in the presence of ConA, this peptide enhanced the ConA induced production of these mediators. In contrast to SIKVAV, the intact laminin molecule failed to influence these monocyte responses. This is the first demonstration that a laminin derived peptide is capable of inducing or enhancing monocyte inflammatory responses that may influence a number of biological activities such as wound healing or excessive connective tissue destruction associated with chronic inflammation.

Amino Acid Sequence↗
PubMed1995

Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model.

BACKGROUND: Angiogenesis is a critical event in wound healing, tumor growth, and the inflammatory vasculitides. Since women have a higher incidence of many vasculitic diseases, we examined the effects of female sex steroids, particularly estradiol, on human umbilical vein endothelial cell (HUVEC) behavior in vitro and on angiogenesis in vivo. METHODS AND RESULTS: HUVECs were grown in estrogen-free medium before each assay. Exogenous 17 beta-estradiol (1 to 5 nmol/L) increased cell attachment to laminin, types I and IV collagen, and fibronectin, as well as to tissue culture plastic. After a confluent monolayer of cells was "wounded" by scraping, estradiol-treated (10(-8) mol/L) cells migrated into the wound three times faster than untreated cells. Cell proliferation on plastic and on laminin increased threefold to fivefold, respectively, in the presence of estradiol. Estradiol also enhanced the ability of HUVECs to organize into tubular networks when plated on a reconstituted basement membrane, Matrigel. Estradiol effects on both the "wounding" assay and tube formation were blocked by the specific estrogen receptor antagonist ICI 182,780. Ovariectomy markedly decreased in vivo vascularization of Matrigel plugs coinjected with basic fibroblast growth factor in mice. With estrogen replacement, angiogenesis was increased to the levels observed in nonovariectomized mice. CONCLUSIONS: These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.

Animals↗
PubMed1995

E-selectin-mediated dynamic interactions of breast- and colon-cancer cells with endothelial-cell monolayers.

The molecular mechanisms involved in the dynamic interaction of human breast carcinoma cells with the endothelial cell lining of lymphatic vessels and post-capillary blood venules are largely unknown. In the present study, laminar flow assays were used to investigate the ability of various normal breast cells and of breast- and colon-tumor cells to adhere to human umbilical cord endothelial cell monolayers. MCF-10A breast, MCF-7 and T-47D breast-carcinoma and clone A, RKO, and HT-29 colon-carcinoma cells accumulated and rolled, in the presence of flow, on tumor necrosis factor (TNF)-stimulated but not on unstimulated endothelial cell monolayers. Non-tumor and tumor cells continued to form transient adhesions with TNF-stimulated endothelial cells even when the flow rate was increased to levels found in arteries. Incubation of TNF-stimulated endothelial cells with an E-selectin-specific monoclonal antibody (MAb) partially or completely inhibited dynamic interactions and diminished adhesion strength, whereas integrin beta 1- and integrin alpha 6-specific MAbs had no effect. A set of highly invasive breast-carcinoma cells (MDA-231, BT-549, HS-578t) neither adhered to nor rolled on resting or TNF-stimulated endothelial cell monolayers. However, after 5 min of static incubation, a fraction of these cells attached strongly to resting and TNF-stimulated endothelial cells and this static adhesion could not be blocked by an E-selectin-specific monoclonal antibody. Our results suggest that E-selectin is a major homing receptor in the metastasis of some breast and colon cancers.

Breast Neoplasms↗
PubMed1995

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.

Capillaries↗