PubMed Health⌕ Search

Biomedical subjects

G Lamson

Publications and source records attributed to G Lamson.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of a new member of the TNF family expressed on antigen presenting cells.

The TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor superfamily member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

Amino Acid Sequence↗
PubMed1993

Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition.

Estrogen receptor-negative Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 as major binding protein (BP) species. Our previous immunohistochemical studies (Oh, Y., Müller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) Endocrinology 131, 3123-3125) have demonstrated the existence of cell surface-associated IGFBP-3 and release of cell surface-associated IGFBP-3 into conditioned media by addition of IGF peptide in Hs578T cells. In this study, we have demonstrated that IGFBP-3 binding on the cell surface is specific and receptor-mediated, by showing: 1) a dose-dependent increase of IGFBP-3 binding by addition of divalent cations (CaCl2 and MnCl2); 2) dose-dependent competition of 125I-IGFBP-3E. coli by unlabeled IGFBP-3E.coli (> 80% competition at 100 nM), but not by IGFBP-1 or fibronectin. In addition, exogenous IGFBP-3 treatment resulted in a significant inhibitory effect on monolayer growth of Hs578T cells. This inhibitory effect of IGFBP-3 was shown to be specific and IGF-independent by demonstrating: 1) dose-dependent inhibition on cell growth (60% inhibition at 20 nM) and inhibition on DNA synthesis (10 nM; p < 0.05, 20 nM; p < 0.005) by exogenous IGFBP-3E. coli, but not by IGFBP-1; 2) absence of stimulatory effects on monolayer cell growth by either native IGFs or IGF analogs which have significantly decreased affinity for IGFBPs, but retain full affinity for type 1 and 2 IGF receptors; 3) significant diminution of the IGFBP-3 inhibitory effects on monolayer growth by coincubation with native IGFs, but not by coincubation with IGF analogs with decreased affinity for IGFBP-3. In conclusion, exogenous IGFBP-3 shows specific binding on the cell surface and can inhibit Hs578T cell monolayer growth by itself, suggesting the existence of specific membrane-associated proteins or receptors for IGFBP-3. Furthermore, IGF-I and -II can attenuate inhibitory effect of IGFBP-3 by forming IGF.IGFBP-3 complexes, thereby preventing cell surface binding of IGFBP-3.

Amino Acid Sequence↗
PubMed1993

Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth.

The insulin-like growth factors (IGFs) are potent mitogens with both endocrine and autocrine-paracrine effects on cell growth. The IGF binding proteins (IGFBPs) are found in many human biological fluids and in the conditioned media of many cell cultures. These molecules are ontogenically and hormonally regulated. Nevertheless, the biological role(s) of the IGFBPs remain controversial. Both inhibitory and stimulatory effects of IGFBPs on cell growth have been suggested. In order to evaluate the actions that endogenously produced IGFBPs have on cell growth, we constructed a mammalian expression vector containing the human IGFBP-3 cDNA and transfected the murine Balb/c cell line. While plasmid-transfected control Balb/c cells (Tx-P) produced only small amounts of a 28-kilodalton IGFBP, the IGFBP-3-transfected Balb/c cells (Tx-BP-3) additionally expressed readily detectable amounts of the characteristic 40- to 44-kilodalton human IGFBP-3 protein doublet and its mRNA. Growth of Tx-BP-3 in serum-containing media was significantly (2.5-fold) slower compared with Tx-P grown under identical conditions. Fully confluent Tx-BP-3 cells arrested their growth at a cell density that was 3-fold lower than did Tx-P cells. When nontransfected cells were grown in the presence of high concentrations of either IGF-I or insulin, growth was equally stimulated. However, when transfected cells were grown in insulin-containing media (5 micrograms/ml), growth rates of the IGFBP-3-transfected cells were not restored to those observed in plasmid-transfected control cells. These results suggest that in this model, the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Transfection of the human IGFBP-3 gene into Balb/c fibroblasts: a model for the cellular functions of IGFBPs.

The insulin-like growth factors (IGFs) are potent mitogens with both endocrine and autocrine-paracrine effects on cell growth. The insulin-like growth factor binding proteins (IGFBPs) are found in many human biological fluids and in the conditioned media of many cell cultures. These molecules are ontogenically and hormonally regulated. Nevertheless, the biological role(s) of the IGFBPs remain controversial. Both inhibitory and stimulatory effects of IGFBPs on cell growth have been suggested. In order to evaluate the actions of endogenously produced IGFBPs on cell growth, we have constructed a mammalian expression vector containing the hIGFBP-3 cDNA, and transfected the murine Balb/c cell line. The IGFBP-3 transfected Balb/c cells (Tx-BP-3) expressed readily detectable amounts of hIGFBP-3 protein and its mRNA. Growth of Tx-BP-3 in serum containing media was significantly slower, compared with control plasmid-transfected cells (Tx-P) grown under identical conditions. Fully confluent Tx-BP-3 cells arrested their growth at a cell density that was lower than did Tx-P cells. When transfected cells were grown in insulin-containing media, growth rates of the IGFBP-3 transfected cells were not restored to those observed in control plasmid-transfected cells. These results suggest that in this model, the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth.

3T3 Cells↗
PubMed1993

Insulin-like growth factor binding protein (IGFBP)-3 levels in conditioned media of Hs578T human breast cancer cells are post-transcriptionally regulated.

Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 (41-kDa and 39-kDa) and IGFBP-4 (24-kDa) as major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by use of cell monolayer affinity cross-linking or by employing immunoperoxidase staining of the cell surface with specific polyclonal anti-human IGFBP-3 antibodies (alpha IGFBP-3gl and alpha IGFBP-3ngl). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media(CM) following addition of IGF-I and IGF-II (maximum 8-13-fold increase at 100 ng/ml concentration), but not by insulin up to 1 microgram/ml; 2) confirmation of IGF-induced increases in CM concentrations of IGFBP-3 by means of Western ligand blot, affinity cross-linking, and IGFBP-3-specific radioimmunoassay; 3) increase of IGFBP-3 in CM by addition of IGF analogs which retain full affinity for IGFBPs ([Leu27]IGF-II and [Tyr55,Gln56]IGF-I), but not by IGF analogs which have significantly decreased affinity for IGFBPs ([Gln3,Ala4,Tyr15,Leu16]IGF-I, [Gln6,Ala7,Tyr18,Leu19,Leu27]IGF-II and IGF-I/insulin hybrid); 4) no change in IGFBP-3 mRNA following addition of IGFs; 5) existence of metal ion-dependent IGFBP-3 specific protease in CM and protection of IGFBP-3 from protease by formation of [IGF:IGFBP-3] complexes; and 6) release of cell surface-associated IGFBP-3 into CM by addition of IGF peptides. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated events, including dissociation of cell surface-associated IGFBP-3 and protection of IGFBP-3 from protease activity.

Blotting, Northern↗
PubMed1992

Insulin-like growth factors (IGFs): implications for aging.

The insulin-like growth factors (IGF)-I and IGF-II are peptides with structural homology to insulin and potent mitogenic and anabolic actions in vitro and in vivo. IGF-I levels are growth hormone (GH)-dependent and vary strikingly with age. IGF-I levels are typically low in infancy and childhood, increase dramatically during puberty, and then gradually decline with advancing age. Whether age-associated changes in GH production or sex steroid secretion, or other unknown factors, cause diminished IGF production in the elderly remains to be determined. In the brain, IGF-II appears to be the most prevalent IGF, but a truncated form of IGF-I also has been recognized. IGF actions are mediated by binding to a family of receptors, which includes the insulin receptor, the structurally homologous type I IGF receptor, and the IGF-II/M-6P receptor, all of which are found in the central nervous system. Additionally, the IGFs bind with high affinity to a family of IGF-binding proteins (IGFBPs). Of the six known IGFBPs, IGFBP-2 appears to be the major one in the mammalian brain and is a major component of CSF. Immunoreactive IGFBP-2 has been identified in astrocytes, and its mRNA has been identified in fetal and adult brain and choroid plexus. The IGFBPs transport the IGFs in serum and other body fluids and appear to regulate IGF access to receptors. In vivo regulation of IGFBPs includes tissue-specific proteases, which cleave specific IGFBPs, altering their affinities for IGF peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Non-receptor mediated, post-transcriptional regulation of insulin-like growth factor binding protein (IGFBP)-3 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells secrete insulin-like growth factor binding protein 3 (IGFBP-3) as the major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by the use of cell monolayer affinity cross-linking or immunoperoxidase staining of the cell surface with a specific polyclonal anti-human IGFBP-3 antibody (alpha IGFBP-3 gamma 1). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media (CM) following addition of IGF-I and -II (maximum 13 fold increase at 100 ng/ml), but not by insulin up to 1 mg/ml; 2) no change in CM IGFBP-3 level by [Gln3,Ala4,Tyr15,Leu16] IGF-I, which has decreased affinity for IGFBPs; 3) no change in IGFBP-3 mRNA following addition of IGFs; 4) release of cell surface-associated IGFBP-3 into CM by the addition of IGFs, but not by [Gln3,Ala4,Tyr15,Leu16]IGF-I. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated dissociation of cell surface-associated IGFBP-3.

Blotting, Northern↗
PubMed1991

Insulin-like growth factor-II (IGF-II) and IGF binding proteins in human endometrium.

The insulin-like growth factor autocrine/paracrine system is believed to play a role in steroid-mediated endometrial differentiation. It is constituted of the mitogenic peptides (IGF-I and IGF-II), membrane receptors, and a family of high-affinity binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. We have investigated expression of the mRNAs encoding the three major IGFBPs (IGFBP-1, IGFBP-2, and IGFBP-3) in human endometrium and have found, by Northern analysis, differential expression of all three mRNAs in secretory compared to proliferative endometrium, different steroidal milieux. IGF-II mRNAs were also detected in secretory endometrium. Finally, we found that human endometrial stromal cells in culture synthesize and secrete IGFBP-2 and IGFBP-3, and that the synthesis of IGFBP-2 is regulated by steroid hormones.

Autoradiography↗
PubMed1991

Insulin-like growth factor binding proteins in human endometrium: steroid-dependent messenger ribonucleic acid expression and protein synthesis.

The insulin-like growth factor (IGF) autocrine/paracrine system is believed to play a role in endometrial differentiation and trophoblast growth. The IGFs bind with high affinity to a family of binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. IGFBP-1, one of three well-characterized IGFBPs in humans, has been shown by numerous investigators to be present in secretory but not proliferative endometrium; however, no information is available with regard to two other human IGFBPs, IGFBP-2 and IGFBP-3, in normal human endometrium. We have examined expression of the messenger RNAs (mRNAs) encoding IGFBP-2 and IGFBP-3 in human proliferative endometrium [under the influence of estradiol (E2)] compared to secretory endometrium (under the influence of E2 and progesterone). Using a complementary DNA probe specific for IGFBP-2, by Northern analysis, expression of a 1.4 kilobase mRNA was found to be differentially expressed in secretory compared to proliferative phase endometrium. Using a complementary DNA probe encoding IGFBP-3, a 2.4 kilobase mRNA was found in endometrium and was also found to be differentially expressed in secretory compared to proliferative phase endometrium. Endometrial stromal cells were established in culture and revealed constitutive synthesis and secretion of IGFBP-2 and IGFBP-3 into the conditioned medium (CM), as detected by Western ligand blot analysis of the CM. Identification of the IGFBPs in the CM was made using IGFBP-2 and IGFBP-3 specific antiserum. In the presence of E2 and progesterone, there was an enhancement in the amount of IGFBP-3 and a marked enhancement of IGFBP-2 in the conditioned media, suggesting sex steroid-dependence of IGFBP-2 and, to a lesser extent, IGFBP-3 protein synthesis. Western ligand blot analysis of IGFBPs in serum throughout the menstrual cycle revealed no hormonal dependence in the serum IGFBPs, suggesting local action of the IGFBPs in endometrium. These data thus show that mRNAs encoding IGFBP-2 and IGFBP-3 are expressed in human endometrium, that their expression is differentially regulated in secretory compared to proliferative phase endometrium (different steroidogenic environments), and that the synthesis of both IGFBP-2 and IGFBP-3 proteins is regulated by steroid hormones.

Autoradiography↗
PubMed1991

A simple assay for proteolysis of IGFBP-3.

An assay for assessing the proteolysis of IGFBP-3 by a biological fluid or protein has been developed using 125IhIGFBP-3 recombinantly derived from Escherichia coli as a substrate indicator. The labelled substrate is incubated at 37 degrees in the presence of the agent being tested and proteolysis is visualized by SDS-PAGE and autoradiography. The assay showed that the pregnancy associated protease is Ca++ dependent. The substrate is also proteolyzed by rat and mouse pregnancy sera, making the assay applicable to studies in those animal systems.

Animals↗
PubMed1991

Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins in primary cultures of prostate epithelial cells.

Insulin-like growth factors (IGFs) are potent mitogens that bind with high affinity and specificity to IGF receptors and IGF-binding proteins (IGFBPs). We studied the roles of these three groups of proteins in prostate epithelial cells (PEC) in primary culture grown under serum-free conditions. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from PEC revealed an abundance of type 1 IGF receptors and no evidence of type 2 IGF receptors. Western ligand blots of conditioned media (CM) from PEC demonstrated the presence of two specific IGFBP bands similar to those previously demonstrated in seminal plasma, with approximate mol wt of 31 and 24 kDa. The 31-kDa band was immunoprecipitable with an antibody to IGFBP-2, and neither band could be deglycosylated with endoglycosidase-F. Northern blot analysis of poly(A)+ RNA prepared from PEC with cDNAs for hIGFBP-1, -2, and -3 documented the expression of mRNA for hIGFBP-2 only. Modifications of the serum-free conditions of PEC did not significantly alter the IGFBP profile of PEC CM. The ability of IGF-I, IGF-II, and insulin to stimulate clonal growth of PEC was examined. IGF-I stimulated PEC growth with an ED50 of 0.1 ng/mL. IGF-II and insulin, respectively, were 1 and 3 orders of magnitude less effective than IGF-I in stimulating the growth of PEC. Radioimmunoassayable IGF-I and IGF-II levels in PEC CM were below the assay detection levels. In conclusion, we suggest that IGFs are important growth stimulators of PEC in culture, that their actions are mediated through the type 1 IGF receptor, and that PEC produce hIGFBP-2 and a 24-kDa IGFBP which may modulate IGF action in these cells.

Blotting, Western↗
PubMed1990

Pituitary control of growth in the neonatal rat: effects of neonatal hypophysectomy on somatic and organ growth, serum insulin-like growth factors (IGF)-I and -II levels, and expression of IGF binding proteins.

The neonatal period is a time of transition between pituitary-independent fetal growth and the pituitary-dependent growth seen in older mammals. To evaluate pituitary-dependent neonatal growth, Wistar rats were hypophysectomized (Hx) on postnatal day 6. Nineteen days post-Hx, body weight and tail length were inhibited 48% and 34%, respectively, compared with sham-Hx controls. Organ weights determined on days 10, 15, 20, 25, and 30 revealed three patterns of pituitary-dependence: 1) pituitary-independent growth in the brain and lung; 2) moderate pituitary-dependent growth in the heart, liver, kidney, and intestine; and 3) marked pituitary-dependent growth in the adrenals, spleen, and testes. Both serum insulin-like growth factor (IGF)-I and -II levels fell significantly in Hx pups by 54 h after Hx (P = 0.0005), and Northern analysis on day 15 showed a significant decrease in liver messenger RNA (mRNA) for IGF-II. Analysis of the major IGF binding proteins (BPs) was performed by Western ligand blots. Hx performed on day 6 resulted in a linear decrease in the amount of the 22k BP from day 10 to day 30. In contrast, the major neonatal BP (IGFBP-2, a 29.5k molecule) showed a biphasic response to neonatal Hx. On postnatal day 10, 4 days after Hx, a significant decrease in IGFBP-2 occurred, which persisted through day 15; by postnatal day 20 and continuing through postnatal day 30, the amount of IGFBP-2 in the serum dramatically increased. The 40 to 50k fraction of IGFBP-3 first appeared in significant quantities by postnatal day 20, and after Hx dropped to 10% of sham-control values. Similarly, Northern analysis on day 15 demonstrated a significant decrease in liver, but not brain, mRNA for IGFBP-2 after Hx, whereas on postnatal day 25, liver mRNA for IGFBP-2 was increased in Hx pups compared with sham controls. We conclude that the pituitary gland exerts significant but selective effects on neonatal growth, with the notable exception of brain growth. Serum levels of both IGF-I and IGF-II, as well as their BPs, are pituitary dependent in the neonatal period. Pituitary-dependent neonatal growth thus appears to be mediated by IGF and modulated by IGF-binding proteins. On the other hand, that portion of the persistent growth in the neonatal Hx rat that is independent of the pituitary-IGF axis may be a good model for investigation of fetal growth.

Adrenal Glands↗
PubMed1990

Identification of insulin-like growth factor-binding protein-2 (IGF-BP-2) and a low molecular weight IGF-BP in human seminal plasma.

Three distinct binding proteins for the insulin-like growth factors (IGF-BPs) have recently had their cDNAs cloned and sequenced. We have evaluated IGF-BP expression in normal, oligospermic and azospermic human seminal plasma (SP), and have identified IGF-BP-2 in all samples. Additionally, SP contains a non-glycosylated low molecular weight IGF-BP (24K), which does not appear to be structurally or immunologically related to IGF-BP-1, -2 or -3.

Base Sequence↗
PubMed1990

Insulin-like growth factor binding proteins in human breast cancer cells: relationship to hIGFBP-2 and hIGFBP-3.

Human breast cancer cells (HBCC) secrete at least four different forms of IGFBPs. We have previously demonstrated that hIGFBP-1 is a minor component of IGFBPs secreted by Hs578T cells and is absent in CM from MCF-7 cells. In our present report, we describe the immunological and structural relationship of HBCC IGFBPs to hIGFBP-2 and hIGFBP-3. Analysis of conditioned media (CM) from Hs578T by Western ligand blotting revealed three IGFBPs of apparent Mr = 38K, 28K, and 24K; CM from MCF-7 revealed only two IGFBPs, of apparent Mr = 31K and 24K. Immunoprecipitation studies with polyclonal antibodies raised against hIGFBP-2 and hIGFBP-3 demonstrated that the 38K IGFBP in Hs578T CM is immunologically related to hIGFBP-3, while the 31K IGFBP in MCF-7 cells is related to the hIGFBP-2. Analysis by Northern blot demonstrated that MCF-7 cells contained mRNA for hIGFBP-2, while Hs578T cells contained the mRNA characteristic of the hIGFBP-3. The identity of the 24K IGFBP remains unknown, and may represent a distinct IGFBP. Of note, assay of CM following removal of BPs by acid chromatography demonstrated no detectable IGF-I or -II. The role of these IGFBPs in HBCC is of interest in view of the potential modulation of IGF actions by these proteins.

Blotting, Western↗