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Biomedical subjects

H K Kleinman

Publications and source records attributed to H K Kleinman.

At least 73 records · Page 4Linked to original sources
PubMed1998

Inhibition of melanoma pulmonary metastasis by methylxanthines due to decreased invasion and proliferation.

Theophylline- and caffeine-treated B16-F10 cells exhibited low adhesion to laminin/collagen type IV and reduced invasion through Matrigel in an in vitro assay. In contrast, theobromine appeared ineffective. When young adult C57BL/6 mice were injected intravenously with theophylline-treated B16-F10 cells, the number of surface lung tumours was markedly reduced. Densitometric analyses performed on digitalized microscopic images of histological sections of lung were used to estimate the frequency (number of lung foci; NLF) and the size (average area of metastatic foci; AMF) of the resulting tumour foci. These parameters were correlated to the proliferation (AMF) and invasion (NLF) of melanoma cells in vivo. The data showed a similar theophylline-induced decrease in the AMF and NLF values (71%, P < 0.01). Caffeine treatment produced a more pronounced decrease in the AMF (61%, P < 0.01) than in the NLF (25%, P < 0.01). To our knowledge, this is the first demonstration that theophylline and caffeine possess the capacity to inhibit not only cell proliferation, but also the metastatic behaviour of melanoma cancer cells.

Animals↗
PubMed1998

Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by curcumin.

Angiogenesis is a crucial step in the growth and metastasis of cancers. Curcumin inhibits tumor initiation and growth. We analyzed the effect of curcumin on endothelial cell migration, attachment, and tube formation on Matrigel. Curcumin had no effect on endothelial cell migration or attachment to either plastic or Matrigel. Curcumin treatment resulted in a dose-dependent inhibition of tube formation when the cells were treated before plating or at the time of plating on Matrigel. Curcumin treatment also caused the preformed tubes to break down. Curcumin inhibited angiogenesis in a s.c. Matrigel plug model in mice. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, zymography was performed to determine whether curcumin affected protease activity. Zymographs of curcumin-treated culture supernatants showed a decrease in the gelatinolytic activities of secreted 53- and 72-kDa metalloproteinases. Western and Northern analysis showed a dose-dependent decrease in the protein expression and transcript of 72 kDa, indicating that curcumin may be exerting its inhibitory effect at both the transcriptional and posttranscriptional level. These findings suggest that curcumin acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis. Curcumin could be developed as an antiangiogenic drug.

Animals↗
PubMed1998

Differential effects of hepatocyte growth factor isoforms on epithelial and endothelial tubulogenesis.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a pleiotropic cytokine that acts as a mitogen, motogen, and morphogen for a variety of cell types. HGF/NK1 and HGF/NK2 are two naturally occurring truncated variants of HGF/SF, which extend from the NH2 terminus through the first and second kringle domain, respectively. Although these variants have been reported to have agonistic or antagonistic activity relative to HGF/SF in assays of cell proliferation and motility, their potential morphogenic activity has not been investigated. To address this issue, we assessed the ability of HGF/NK1 and HGF/NK2 to induce tube formation by (a) MCF-10A mammary epithelial cells grown within collagen gels and (b) human umbilical vein endothelial (HUVE) cells grown on Matrigel. We found that HGF/NK1 stimulated tubulogenesis by both MCF-10A and HUVE cells, whereas HGF/NK2 did not stimulate tubulogenesis, but efficiently antagonized the morphogenic effect of full-length HGF/SF. HGF/NK1 and HGF/NK2 also had agonistic and antagonistic effects, respectively, on MCF-10A cell proliferation and HUVE cell migration. These results demonstrate that HGF/NK1, which only consists of the NH2-terminal hairpin and first kringle domain, is sufficient to activate the intracellular signaling pathways required to induce morphogenic responses in epithelial and endothelial cells. In contrast, HGF/NK2, which differs from HGF/ NK1 by the presence of the second kringle domain, is devoid of intrinsic activity but opposes the effects of HGF/SF. The differential properties of the two HGF/SF isoforms provide a basis for the design of more potent HGF/SF agonists and antagonists.

Cell Division↗
PubMed1997

Identification of cell binding sequences in mouse laminin gamma1 chain by systematic peptide screening.

Laminin-1, a major component of basement membranes, consists of three different chains designated alpha1, beta1, and gamma1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin gamma1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the gamma1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-alpha2beta1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin gamma2 chain. These results suggest that the specific sequences on the laminin gamma1 chain are biologically active and interact with distinct cell surface receptors.

Amino Acid Sequence↗
PubMed1997

Laminin chain expression by chick chondrocytes and mouse cartilaginous tissues in vivo and in vitro.

We have observed that laminins are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and adult mouse knee joint by the methods of in situ hybridization, immunohistochemistry, Western blotting, and immunoprecipitation. From in situ hybridization using similar sized RNA probes for different mouse laminin chains, mRNAs for the alpha 1, alpha 2, beta 1, beta 2, and gamma 1 chains were expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and the articular cartilage cap and epiphyseal growth plate of adult mouse knee joint. Through the use of chain-specific antibodies, staining for laminins was observed in the cytoplasm of chondrocytes from chick embryo sternum, mouse limb bud, and adult mouse knee joint. Western blot analysis confirmed the presence of laminin chains in the cells and sternal tissues. Cultured chick embryonic sternal chondrocytes expressed laminin mRNAs in the proliferating stage (2-3 days of culture) but the level increased in the aggregated cells during the maturation stage (5-7 days of culture). Comparable data were also obtained after immunostaining the cells. Thus, laminins are expressed in significant amounts by chondrocytes and may have an important role in cartilage development.

Animals↗
PubMed1997

Liver metastasis formation by laminin-1 peptide (LQVQLSIR)-adhesion selected B16-F10 melanoma cells.

Laminin-1, a major basement membrane glycoprotein, promotes tumor cell malignancy. Incubation of B16-F10 melanoma cells with a peptide containing an active sequence in laminin-1, designated AG-73 (leu-glu-val-glu-leu-ser-ile-arg; LQVQLSIR), enhances in vitro adhesion, migration, invasion and gelatinase production and in vivo lung colonization and metastases to the liver. In the current study, we have tried to define the mechanism of enhancement of liver metastases induced by AG-73 using B16-F10 murine melanoma cells selected for adhesion on AG-73-coated dishes. Cells were sequentially selected for adhesion more than 30 times and then characterized. AG-73 selected cells had much longer cytoplasmic processes and occasionally formed nodular aggregates. AG-73 selected cells attached 1.2- to 1.5-fold better to both AG-73 and laminin-1, were able to invade through the Matrigel-coated filter up to 6-fold more, grew s.c. 1.5-2 times faster, produced twice the number of lung colonies, and showed more liver nodules (12 of 28 vs. 1 of 27) than parental cells. Our data demonstrate that the enhanced malignant phenotype of B16-F10 cells can be observed in the absence of added peptide with the adhesion-selected cells.

Amino Acid Sequence↗
PubMed1997

Regulation of capillary formation by laminin and other components of the extracellular matrix.

The process of angiogenesis (vessel formation) and the resulting stabilization of the mature vessel are complex events that are highly regulated and require signals from both serum and the extracellular matrix. Endothelial cells rest on a specialized thin extracellular matrix known as the basement membrane. Endothelial cells lining normal blood vessels are usually quiescent. When a proper stimulus is present, angiogenesis beings when endothelial cells degrade their basement membrane and invade the surrounding extravascular matrix. Formation of new vessels involves the migration and proliferation of cells. To assist the cells in their migration, the extravascular matrix provides an environment rich in stromal collagen fibers, fibrin, hyaluoronic acid, vitronectin and fibronectin. Once the endothelial cells assemble to form a new vessel, the cells secrete a basement membrane that helps to stabilize and maintain the vessel wall. The basement membrane adheres tightly to cells comprising the vessel wall, provides inductive signals, and plays a important role in the homeostasis of new vessels. We have demonstrated that two major components of the basement membrane, laminin and collagen IV, possess endothelial cell binding sites which regulate vessel stability. In this chapter, we will define the role of these molecules in endothelial cell behavior.

Amino Acid Sequence↗
PubMed1997

Neuronal laminins and their cellular receptors.

The laminins are a family of extracellular matrix glycoproteins expressed throughout developing neural tissues. The laminins are potent stimulators of neurite outgrowth in vitro for a variety of cell types, presumably reflecting an in vivo role in stimulating axon outgrowth. In recent years, the laminins have been shown to occur in several distinct isoforms; currently, the precise functional differences between the laminin variants are not well understood. A variety of neuronal surface receptors have been identified for one laminin isoform, laminin-1. These receptors include several members of the integrin family, as well as non-integrin laminin-binding proteins such as LBP-110, the 67 kDa laminin-receptor, alpha-dystroglycan, and beta 1,4 galactosyltransferase. Little is currently known about receptors for other laminin isoforms.

Animals↗
PubMed1997

Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18.

Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell characteristics, and the malignant RWPE-2 cells, provide useful cell culture models for studies on prostate growth regulation and carcinogenesis.

Androgens↗
PubMed1997

Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells.

Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.

3T3 Cells↗
PubMed1997

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells.

Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.

Cell Movement↗
PubMed1997

Synthesis and activity of partial retro-inverso analogs of the antimetastatic laminin-derived peptide, YIGSR-NH2.

This paper describes the synthesis and biological evaluation of six partial retro-inverso peptidomimetic analogs of YIGSR-NH2, a synthetic peptide from the beta 1 chain of laminin, which has antimetastatic activity. The intent was to improve the antimetastatic potency of YIGSR-NH2 by limiting the in vivo enzymatic degradation through the incorporation of fraudulent peptide bonds. We have prepared the following retro-inverso peptides, Tyr-Ile-Gly-Ser-gArg-CHO (1), Tyr-gIle-mGly-Ser-Arg-NH2 (2), Tyr-gIle-mGly-Ser-gArg-CHO (3), gTyr-D-rIle-mGly-Ser-Arg-NH2 (4), Tyr-Ile-Gly-gSer-D-rArg-CHO (5) and Tyr-gIle-rGly-D-rSer-D-rArg-CHO (6). In vitro assays for B16F10 melanoma cell adhesion showed no significant activity for these six peptides. Peptides 1-3, 5 and 6 were further tested, in vivo, for their ability to inhibit tumor metastases to the lung in mice injected in the tail vein with B16F10 melanoma cells. All five of the retro-inverso peptides tested showed statistically significant inhibition of metastasis, but the most active peptides were 5 and 6, which showed 57 and 69% inhibition of metastasis, respectively.

Animals↗
PubMed1997

Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo.

We have tested the effect of thrombin on endothelial cell tube formation in vitro and angiogenesis in vivo. Thrombin induces the differentiation of endothelial cells into capillary structures in a dose-dependent fashion (0.1-0.3 units thrombin/ml) on Matrigel, a laminin-rich reconstituted basement membrane matrix. At higher thrombin concentrations (1.0 unit/ml), a suppression of tube formation is evident, probably due to downregulation (desensitization) of the thrombin receptor. D-Phe-Pro-Arg-CH2Cl-thrombin is without effect when used alone, but it abolishes the tube-promoting effect of thrombin when used in combination with thrombin, indicating the involvement of the catalytic site of thrombin. Activation of protein kinase C (PKC) seems to be the transduction mechanism involved in the stimulation of tube formation by thrombin. Ro-318220 (3 micrograms/ml), a specific inhibitor of PKC, completely abolishes the stimulatory effect of thrombin. In the in vivo Matrigel system of angiogenesis, there is a 10-fold increase in endothelial cell infiltration in response to thrombin. These results provide evidence for the angiogenesis-promoting effect of thrombin in vivo and the induction by thrombin of the angiogenic phenotype of endothelial cells in vitro in the absence of other cell types such as smooth muscle cells, pericytes, and inflammatory cells.

Animals↗
PubMed1996

Active peptides from the carboxyl-terminal globular domain of laminin alpha2 and Drosophila alpha chains.

The laminin alpha1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the alpha1 chain are conserved in the corresponding regions of the different laminin alpha chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin alpha2 chain and Drosophila laminin alpha chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin alpha2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin alpha1 and alpha2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.

Amino Acid Sequence↗
PubMed1996

Identification of synthetic peptides derived from laminin alpha1 and alpha2 chains with cell type specificity for neurite outgrowth.

Laminin-1 is a potent promoter of neurite outgrowth. Here we find that the laminin-1 alpha chain contains peptide sequences that stimulate attachment and neurite outgrowth in some but not all neuronal cells. Testing of a series of overlapping peptides for biological activity with three laminin-1-responsive, neuronally derived cell lines resulted in the identification of peptide sequences, RKRLQVQLSIRT and KNRLTIELEVRT, from corresponding regions of the alpha1 and alpha2 (merosin) chain globular regions, respectively, that were active for attachment for all three cell lines and for neurite outgrowth with two of the cell lines. These data represent the identification of novel peptide sequences active with certain neuronal cells from the a1 and a2 chains of laminin. The peptides showed different attachment and neurite outgrowth activities depending on the cell type, suggesting cell type specificity in the response of the cells.

Amino Acid Sequence↗
PubMed1996

Expression of an estrogen receptor by human coronary artery and umbilical vein endothelial cells.

BACKGROUND: Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [3H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2 x 10(4) to 8 x 10(4) receptors per cell and a Kd of approximately 5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. CONCLUSIONS: These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.

Arteries↗
PubMed1996

TGF-beta and basement membrane matrigel stimulate the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria.

Primary cultures of fetal rat calvarial cells contain a spectrum of osteogenic phenotypes including undifferentiated mesenchymal cells, osteoprogenitor cells, and osteoblasts. We recently demonstrated that rat calvarial osteoblast-like cells grown on basement membrane undergo profound morphological changes resembling a canalicular network in bone. In the present study, we examined the effect of reconstituted basement membrane Matrigel on chondroblastic versus osteoblastic differentiation of different cell subpopulations obtained by five consecutive enzymatic digestions of rat calvarial cell populations. We found that the appearance of canalicular cell processes decreased with the later digests. When cells from the fourth and fifth digest were grown on top of Matrigel for 7 days, the majority of the cell aggregates displayed chondrocytic characteristics but none of the cells became hypertrophic. When individual chondroblastic cell aggregates were subsequently transferred from Matrigel to plastic, they started expressing types I and X collagens, alkaline phosphatase, and osteocalcin. Within the next 7 days (days 8-14 of the experiment), the majority of cells increased in size, and at day 17 on plastic (day 24 of the experiment) mineralized bone nodules formed. The chondroblastic differentiation of calvarial cells grown on Matrigel could be inhibited by a specific transforming growth factor-beta 1 (TGF-beta 1) but not by a TGF-beta 2 antibody. Addition of recombinant TGF-beta 1 to similar cultures promoted the appearance of chondroblastic cell aggregates. The cartilage phenotype could not, on the contrary, be promoted by growing the cells on other extracellular matrices such as a collagen I gel. We suggest that TGF-beta 1 in concert with the basement membrane extracellular matrix induces chondroblastic differentiation of rat calvarial osteoprogenitor cells.

Alkaline Phosphatase↗