Search PubMed⌕ Search

Biomedical subjects

H K Kleinman

Publications and source records attributed to H K Kleinman.

At least 19 recordsLinked to original sources
PubMed2002

Laminin gamma 1 chain peptide, C-16 (KAFDITYVRLKF), promotes migration, MMP-9 secretion, and pulmonary metastasis of B16-F10 mouse melanoma cells.

Laminin-1, a heterotrimer of alpha 1, beta 1, and gamma 1 chains specific to basement membrane, promotes cell adhesion and migration, proteinase secretion and metastases of tumour cells. Several active sites on the alpha 1 chain have been found to promote B16-F10 melanoma lung colonisation and here we have determined whether additional tumour promoting sites exist on the beta 1 and gamma 1 chains. Recently, we have identified novel cell adhesive peptides derived from laminin beta 1 and gamma 1 chains by systematic screening of synthetic peptides. Nine beta 1 peptides and seven gamma 1 peptides active for cell adhesion were tested for their effects on experimental pulmonary metastases of B16-F10 mouse melanoma cells in vivo. The most active adhesive peptide derived from the gamma 1 chain globular domain, C-16 (KAFDITYVRLKF), significantly enhanced pulmonary metastases of B16-F10 cells, whereas no other peptides showed enhancement. C-16 also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro. Furthermore, C-16 significantly induced the production of MMP-9 from B16-F10 cells. These results suggest that this specific laminin gamma 1 chain peptide has a metastasis-promoting activity and might be a new molecular target of anti-cancer treatment.

Animals↗
PubMed2002

Biological response of chondrocytes to hydrogels.

Primary bovine chondrocytes were encapsulated in alginate and alginate combined with cartilage matrix extract, Cartrigel, for the purpose of cartilage tissue engineering. The cell constructs were incubated in vitro and gene expression of cartilage-specific extracellular matrix molecules was quantitated and localized with in situ hybridization with a decrease in expression observed in the alginate-Cartrigel constructs. Further understanding of cell response to scaffolds will allow rational design and development of hydrogels for cartilage tissue engineering.

Alginates↗
PubMed2001

Identification of homologous biologically active sites on the N-terminal domain of laminin alpha chains.

Laminin, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha, beta, and gamma chains. To date, five different alpha chains have been identified. N-terminal domain VI in the alpha1 chain has various biological activities. Here we screened biologically active sequences on domain VI of the laminin alpha2, alpha3, and alpha5 chains using a large number of overlapping peptides. HT-1080 human fibrosarcoma cell attachment to the peptides was evaluated using peptide-coated plastic plates and peptide-conjugated Sepharose beads. We identified four cell adhesive sequences from laminin alpha2 chain domain VI, two sequences from the alpha3 chain, and two sequences from the laminin alpha5 chain. Sequences homologous to A13 (RQVFQVAYIIIKA, alpha1 chain 121-133) on all the alpha chains (FQIAYVIVKA, alpha2 chain 130-139; GQLFHVAYILIKF, alpha3 chain 96-108; FHVAYVLIKA, alpha5 chain 74-83) showed strong cell attachment activity. A5-16 (LENGEIVVSLVNGR, alpha5 chain 147-160) showed the strongest cell attachment activity in the plate assay, and the homologous peptide in the alpha3 chain promoted similar strong cell attachment activity. A5-16 and its homologous peptide in the alpha2 chain promoted moderate cell attachment, while the homologous peptide to A5-16 in the alpha1 chain did not show activity. A2-7 (SPSIKNGVEYHYV, alpha2 chain 108-120) showed cell attachment activity only in the plate assay, but homologous sequences in the alpha1, alpha3, and alpha5 chains did not promote activity. A2-7 promoted endothelial cell sprouting from aortic rings in vitro and melanoma colonization to murine lungs in vivo. However, none of the homologous peptides of A2-7 promoted experimental pulmonary metastasis by B16-BL6 melanoma cells. These results indicate that there are chain-specific active sites in domain VI of the laminin alpha chains, some of which contain conserved activities.

Amino Acid Sequence↗
PubMed2001

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model.

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

Animals↗
PubMed2001

The role of alpha 6 beta 1 integrin and EGF in normal and malignant acinar morphogenesis of human prostatic epithelial cells.

Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

Antibodies↗
PubMed2001

Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells.

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.

Allantois↗
PubMed2001

Stimulation of tumor growth by human soluble intercellular adhesion molecule-1.

Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance.

Adenocarcinoma↗
PubMed2001

Cell type-specific differences in glycosaminoglycans modulate the biological activity of a heparin-binding peptide (RKRLQVQLSIRT) from the G domain of the laminin alpha1 chain.

AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha1 chain, has diverse biological activities with different cell types. The heparan sulfate side chains of syndecan-1 on human salivary gland cells were previously identified as the cell surface ligand for AG73. We used homologous peptides from the other laminin alpha-chains (A2G73-A5G73) to determine whether the bioactivity of the AG73 sequence is conserved. Human salivary gland cells and a mouse melanoma cell line (B16F10) both bind to the peptides, but cell attachment was inhibited by glycosaminoglycans, modified heparin, and sized heparin fragments in a cell type-specific manner. In other assays, AG73, but not the homologous peptides, inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and truncations. Fewer residues were critical for inhibiting branching morphogenesis (XKXLXVXXXIRT) than those required to inhibit B16F10 network formation on Matrigel (N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a beta-sheet with the N-terminal K (Lys(2)) and the C-terminal R (Arg(10)) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.

Amino Acid Sequence↗
PubMed2001

Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells.

BACKGROUND: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. METHODS: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. RESULTS: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway. CONCLUSIONS: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis.

Antibodies↗
PubMed2001

Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression.

BACKGROUND: The study of prostate carcinogenesis and tumor progression is made difficult by the lack of appropriate in vitro and in vivo models. High prevalence of prostatic intra-epithelial neoplasia and latent prostatic carcinoma, representing multiple steps in carcinogenesis to invasive carcinoma, are relevant targets for cancer prevention. From the RWPE-1, immortalized, non-tumorigenic, human prostate epithelial cell line, we have derived four tumorigenic cell lines with progressive malignant characteristics. METHODS: Cell lines were derived by exposure of RWPE-1 to N-methyl-N-nitrosourea (MNU), selected and cloned in vivo and in vitro, and characterized by prostatic epithelial and differentiation markers, karyotype analysis, anchorage-independent growth, invasiveness, tumorigenicity, and pathology of the derived tumors. RESULTS: Cytokeratins 8 and 18, androgen receptor, and prostate-specific antigen expression in response to androgen, confirm prostatic epithelial origin. RWPE-1 cells do not grow in agar and are not tumorigenic in mice, but the growth, tumorigenicity, and tumor pathology of the MNU cell lines correlate with their invasive ability. The WPE1-NA22 (least malignant) form small, well-differentiated, and WPE1-NB26 cells (most malignant) form large, poorly differentiated, invasive tumors. Overall, loss of heterozygosity for chromosomes 7q, 13q, 18q, and 22, and gain of 5, 9q, 11q, and 20, was observed. The MNU cell lines, in order of increasing malignancy are; WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26. CONCLUSIONS: This family of cell lines with a common lineage represents a unique and relevant model which mimics stages in prostatic intra-epithelial neoplasia (PIN) and progression to invasive cancer, and can be used to study carcinogenesis, progression, intervention, and chemoprevention.

Alkylating Agents↗
PubMed2001

An angiogenic laminin site and its antagonist bind through the alpha(v)beta3 and alpha5beta1 integrins.

Angiogenesis is important for wound healing, tumor growth, and metastasis. Endothelial cells differentiate into capillary-like structures on a laminin-1-rich matrix (Matrigel). We previously identified 20 angiogenic sites on laminin-1 (alpha1beta1gamma1) by screening 559 overlapping synthetic peptides. C16, the most potent gamma1 chain peptide, blocked laminin-1-mediated adhesion and was the only gamma1 chain peptide to block attachment to both collagen I and fibronectin. This suggested that C16 was acting via a receptor common to these substrates. We demonstrated that C16 is angiogenic in vivo. Affinity chromatography identified the integrins alpha5beta1 and alpha(v)beta3 as surface receptors. Blocking antibodies confirmed the role of these receptors in C16 adhesion. C16 does not contain an RGD sequence and, as expected, an RGD-containing peptide did not block C16 adhesion nor did C16 act via MAP kinase phosphorylation. Furthermore, we identified a C16 scrambled sequence, C16S, which antagonizes the angiogenic activity of bFGF and of C16 by binding to the same receptors. Because the laminin gamma1 chain is ubiquitous in most tissues, C16 is likely an important functional site. Since the biological activity of C16 is blocked by a scrambled peptide, C16S may serve as an anti-angiogenic therapeutic agent.

Amino Acid Sequence↗
PubMed2001

The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue.

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Animals↗
PubMed2001

Role of laminin in ovarian cancer tumor growth and metastasis via regulation of Mdm2 and Bcl-2 expression.

Ovarian cancer is among the most lethal cancers in women because of its high metastatic potential and lack of response to therapy. An experimental model to study this disease was developed using a transformed granulosa cell line expressing a mutant p53 and Ha-ras. When injected into the ovary of nude mice in the presence of laminin-1, tumors develop in the ovary and peritoneum and metastasize to various organs, leading to death within 21 days. In contrast, when cells were injected in the presence of gelatin, development of tumors was slower and no metastases were observed by day 21. Here we investigated the possible mechanism by which laminin-1 exerts its promotion of tumorigenesis and metastasis. Cells were co-injected with laminin-1 and active laminin peptides from the alpha1; (A13: RQVFQVAYIIIKA, A12: WVTVTLDL RQVFQ, AG73: LQVQLSIR, IKVAV) and beta1 (YIGSR) chains. Ovarian tumor growth and metastasis were increased in the presence of laminin-1 plus either AG73 peptide, IKVAV, or A13, and were significantly reduced in the presence of A12 or YIGSR. Expression of Bcl-2 and Mdm2 was higher by 3.5- and about 100-fold, respectively, in ovarian tumors grown in the presence of laminin compared to tumors grown in the presence of gelatin. Moreover, peptides A13 and AG73 further elevated Bcl-2 expression by 6- and 7-fold respectively, while IKVAV yielded expression similar to laminin-1. YIGSR and A12 reduced the expression of Bcl-2 by 7- and 3-fold, respectively, compared to treatment with laminin-1. A13 and AG73 increased Mdm2 expression by 1.8- and 1.3-fold, respectively, while IKVAV, A12, and YIGSR were without effect. Thus, laminin-1 exerts its proliferative effect on the development of ovarian tumors via upregulation of survival genes such as Bcl-2 and Mdm2. Peptides A13 and AG73 (which increased tumor growth and spread) enhance the expression of these genes and A12 and YIGSR (which decrease tumor growth and spread) attenuate their expression. IKVAV probably enhances tumor growth and metastasis by another mechanism.

Animals↗
PubMed2001

Role of basement membrane in tumor growth and metastasis.

The basement membrane is a thin extracellular matrix produced by epithelial and endothelial cells. It is biologically active for normal epithelial cell differentiation. Basement membrane promotes the growth of tumor cells in vitro and in vivo when coinjected. Laminin, the major biologically active component, also increases tumor growth and the malignant phenotype by promoting increased cell growth and protease activity. Using systematic peptide screening with synthetic peptides covering the entire laminin molecule, several active sites in laminin have been identified that regulate tumor growth and metastasis.

Basement Membrane↗
PubMed2000

Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells.

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.

Animals↗
PubMed2000

New thiol and sulfodiimine metalloproteinase inhibitors and their effect on human microvascular endothelial cell growth.

Matrix metalloproteinases (MMPs, matrixins) are a family of homologous zinc endopeptidases that may play a very important role in many physiological and pathological processes, e.g., the initiation of angiogenesis. Two new matrixin inhibitors were synthesized and characterized. A thiol inhibitor MAG-283 had IC(50) values of 480, 3, 280, 14, 1.1, and 2.3 nM against human interstitial collagenase (MMP-1), gelatinase A (MMP-2), stromelysin (MMP-3), matrilysin (MMP-7), neutrophil collagenase (MMP-8), and gelatinase B (MMP-9), respectively. A sulfodiimine inhibitor YLL-224 had IC(50) values of 180, 63, 4500, 210, 5.9, and 44 nM against MMP-1, -2, -3, -7, -8, and -9, respectively. Human skin microvascular endothelial cells were treated with these two compounds in culture. These inhibitors at very low micromolar concentrations suppressed proliferation of the endothelial cells stimulated by acidic fibroblast growth factor and vascular endothelial growth factor. They also partially blocked cell invasion through type IV collagen. These results suggested a correlation between the anti-metalloenzyme activity and the effects of these inhibitors on the growth and invasion of endothelial cells.

Amides↗