Biomedical subjects
N Bouck
Publications and source records attributed to N Bouck.
Tumour suppressor genes and angiogenesis: the role of TP53 in fibroblasts.
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A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.
At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.
The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis.
Proliferin stimulates endothelial cell migration in culture and neovascularization in vivo. Previous studies have demonstrated that proliferin can bind to the insulin-like growth factor II/mannose 6-phosphate receptor, and that binding can be blocked by mannose 6-phosphate. We have now found that this receptor plays an essential role in proliferin-induced angiogenesis. Proliferin binding to endothelial cells is blocked by the addition of mannose 6-phosphate, as is the ability of both recombinant and placental-derived proliferin to stimulate the migration of capillary endothelial cells in vitro and to induce neovascularization in the rat cornea. Consistent with a direct role of this receptor in angiogenesis, insulin-like growth factor II, as well as a mutant form of insulin-like growth factor II that binds to the insulin-like growth factor II/mannose 6-phosphate receptor but not to the insulin-like growth factor I receptor, also stimulate endothelial cell migration and neovascularization.
Inhibition of angiogenesis by thrombospondin-2.
To assess the ability of proteins of the thrombospondin family to inhibit angiogenesis, recombinant murine thrombospondin-2, bovine thrombospondin-2/CISP and thrombospondin-5/COMP were purified and tested for ability to block the migration of capillary endothelial cells towards a variety of inducers and to inhibit neovascularization induced in the rat cornea. Both preparations of thrombospondin-2 were active inhibitors in vitro and in vivo whereas thrombospondin-5/COMP was inactive. These results define thrombospondin-2 as a newly identified naturally occurring inhibitor of angiogenesis and suggest that the properdin-like type 1 modules that it shares with antiangiogenic thrombospondin-1 and are missing in thrombospondin-5/COMP could contribute to this activity.
The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression.
Fifteen different natural inhibitors of angiogenesis have now been identified that are produced by mammalian cells and are able to block in vivo neovascularization. The majority of these are able to inhibit endothelial cell activities in vitro and all those tested have demonstrated significant antitumor activity. Most normal cells produce inhibitors of neovascularization that must be downregulated before the cells can develop into angiogenic, malignant tumors. In several cases the production of inhibitors ceases when tumor suppressor genes are inactivated. In the BT549 human breast carcinoma cell line, the reintroduction of a wild type p53 tumor suppressor gene resulted in the stimulation of the secretion of an inhibitor of angiogenesis, thrombospondin-1, and as a result the cells lost their angiogenic phenotype and became able to suppress angiogenesis induced by the parental tumor line. These results provide a new example of tumor suppressor gene control of a natural inhibitor of angiogenesis and add support to the concept that thrombospondin loss may play an important role in the development of some human breast cancers.
Stimulation and inhibition of angiogenesis by placental proliferin and proliferin-related protein.
In many mammalian species, the placenta is the site of synthesis of proteins in the prolactin and growth hormone family. Analysis of two such proteins, proliferin (PLF) and proliferin-related protein (PRP), revealed that they are potent regulators of angiogenesis; PLF stimulated and PRP inhibited endothelial cell migration in cell culture and neovascularization in vivo. The mouse placenta secretes an angiogenic activity during the middle of pregnancy that corresponds primarily to PLF, but later in gestation releases a factor that inhibits angiogenesis, which was identified as PRP. Incubation of placental tissue with PLF led to the specific binding of this hormone to capillary endothelial cells. Thus PLF and PRP may regulate the initiation and then the cessation of placental neovascularization.
Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1.
As normal cells progress toward malignancy, they must switch to an angiogenic phenotype to attract the nourishing vasculature that they depend on for their growth. In cultured fibroblasts from Li-Fraumeni patients, this switch was found to coincide with loss of the wild-type allele of the p53 tumor suppressor gene and to be the result of reduced expression of thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis. Transfection assays revealed that p53 can stimulate the endogenous TSP-1 gene and positively regulate TSP-1 promoter sequences. These data indicate that, in fibroblasts, wild-type p53 inhibits angiogenesis through regulation of TSP-1 synthesis.
Inhibition of angiogenesis by tissue inhibitor of metalloproteinase.
Matrix proteases play a critical role in cell invasion and migration, including the process of angiogenesis. The ability of specific factors to induce angiogenic responses correlates with their stimulation of matrix protease synthesis and release. Using an in vivo angiogenesis assay, the endothelial cell response to known angiogenic factors, basic fibroblast growth factor (bFGF) and adipocyte conditioned medium, was blocked by an inhibitor of matrix metalloproteinase activity, TIMP-1. The TIMP effect was mediated, at least in part, through the inhibition of endothelial cell migration, as determined by the ability of TIMP to block chemotaxis in a Boyden chamber assay. These results indicate that the inhibition of migration is a direct effect on the endothelial cells and does not require accessory cells. An additional observation was that the RNA levels for TIMP were significantly reduced in differentiated adipocytes, compared to undifferentiated F442A controls. Therefore, the acquisition of an angiogenic phenotype may involve not only the induction of positive factors, but also the suppression of angiogenesis inhibitors.
The p53 tumor suppressor gene inhibits angiogenesis by stimulating the production of thrombospondin.
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Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.
Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2-terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19-residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.
Angiogenesis: a mechanism by which oncogenes and tumor suppressor genes regulate tumorigenesis.
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Tumor angiogenesis: the role of oncogenes and tumor suppressor genes.
The progressive growth of solid tumors is strictly dependent on their ability to attract new blood vessels that will supply them with oxygen and essential nutrients. Although in experimental models a number of compounds are able to elicit such angiogenesis, under normal physiological conditions new vessel growth is tightly repressed. One consequence of the cascade of oncogene activation and tumor suppressor gene loss that produces a solid tumor must be the alleviation of this normal repression. Understanding how this is achieved may help to define the functions of oncogenes and suppressor genes that are relevant to tumorigenicity and may suggest novel therapeutic strategies.
Interactions between hyperthermia and irradiation in two human lymphoblastic leukemia cell lines in vitro.
Thermal radiosensitization was studied in two human T-cell acute lymphoblastic leukemia cell lines (JM and MOLT3) with regard to heat-irradiation sequence and heating duration. In MOLT3 thermal radiosensitization was maximal when 43.5 degrees C hyperthermia immediately preceded or followed irradiation; at 41.5 degrees C, radiosensitization was maximal with hyperthermia immediately before or up to 3 h after irradiation. In JM, enhancement of radiation killing was unexpectedly maximal when 41.5 or 43.5 degrees C hyperthermia preceded irradiation by 2 to 4 h. Thermal radiosensitization increased exponentially with increasing duration of heating at 41.5 degrees C for at least 3 h in MOLT3. In contrast, in JM, radiosensitization increased exponentially for 1.6 h but additional heating (up to 3 h net heating) had no appreciable further effect on radiation killing. For JM, repair of single and double stranded DNA breaks was investigated using alkaline and neutral elution techniques to determine whether the unusual results regarding heat-irradiation sequencing were related to effects of heat on repair of DNA damage. These studies were unable to detect significant differences in repair of single or double stranded DNA breaks between unheated control cells and cells heated at 41.5 degrees C for 1 h ending 4 h before irradiation. The direct cytotoxicity of hyperthermia was also studied in both cell lines.
Genetic assessment of the strength of a cancer suppressor gene in hamster cells.
Tetraploid and near-tetraploid chemically transformed derivatives of the pseudodiploid hamster line BHK 21/clone 13 were prepared in four different ways and their ability to be suppressed for anchorage independence by fusion to the anchorage-dependent parental line was tested. In all cases the presence of a single normal genome, thought on genetic grounds to contain a single suppressor gene, was able to prevent the anchorage-independent growth of transformed lines whether they contained one or two complements of pseudodiploid chromosomes. Suppression of the single step in carcinogenesis that is registered by BHK cells as they transform to anchorage independence is thus unusually powerful and apparently independent of chromosome balance and of strict dosage effects.
Phagocyte-mediated carcinogenesis: DNA from phagocyte-transformed C3H 10T1/2 cells can transform NIH/3T3 cells.
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Coordinate control of anchorage independence, actin cytoskeleton, and angiogenesis by human chromosome 1 in hamster-human hybrids.
A panel of hybrids previously derived from fusions between a chemically transformed hamster cell line and normal human fibroblasts (A. Stoler and N. Bouck, Proc. Natl. Acad. Sci. USA, 82: 570-574, 1985) has been used to test whether or not anchorage independence, lack of actin cables, and angiogenic activity, three characteristics of transformed cells considered necessary but not sufficient for neoplasia, are coordinately regulated. In these hybrids anchorage independence is initially suppressed and those hybrids where it remains suppressed have been shown to retain human chromosome 1. Here we show that suppressed hybrids also display actin microfilament cables characteristic of normal cells and are unable to elicit an angiogenic response in the rat cornea assay. In contrast, those hybrids in which anchorage independence is expressed and which have lost human chromosome 1 have an actin cytoskeleton resembling that of the transformed parent and are potently angiogenic.
The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity.
Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus.