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Blood-Clotting Inhibitor Prevents Angiogenesis, Tumor GrowthA team of researchers led by Judah Folkman, the Julia Dyckman Andrus professor of pediatric surgery at Children's Hospital, has discovered that certain forms of a naturally occurring anticoagulant, antithrombin, have potent anti-angiogenic and antitumor activity. The study by Folkman, first author Michael O'Reilly, clinical fellow in radiation oncology at the Joint Center for Radiation Therapy, and colleagues is published in the Sept. 17 Science and provides further evidence that blood-clotting pathways play a key role in the regulation of angiogenesis. The specific form of antithrombin found to have these properties is the structure that forms upon cleavage of the carboxyl-terminal loop. This cleaved molecule was isolated from a small-cell lung cancer cell line after being screened for its ability to generate factors that inhibit endothelial cell proliferation and tumor growth (see figure). Both this form, and the uncleaved inactive (latent) form, inhibited capillary endothelial cell proliferation induced by growth factors. The cleaved and latent forms of antithrombin are similar in structure. The researchers then tested whether these compounds could inhibit tumor growth in mice. Subcutaneous injection of either the cleaved or latent forms of antithrombin potently inhibited tumor growth and caused the tumors to regress to small subcutaneous nodules.
A tumor variant implanted in mice (top) makes a form of antithrombin that inhibits growth of a second implanted tumor (arrows). A second variant does not make this substance and shows no tumor inhibition (bottom). Growth Factor May Prevent Spread of Gallbladder CancerTransforming growth factor (TGF)-beta1, an intracellular mediator with multiple functions, is highly expressed in several cancers and is often associated with increased metastatic capacity and poor prognosis. It is not known, however, whether the growth factor has any direct role in the metastatic activity. HMS researchers at Massachusetts General Hospital have discovered that TGF-beta1 is expressed in a wide variety of human gallbladder tumors and, surprisingly, release of this molecule from primary gallbladder tumors in mice actually suppresses angiogenesis of tumors at a distant site. Apparently, the microenvironment of the gallbladder tumor plays a key role in TGF-beta1 production. The study from the laboratory of Rakesh Jain, the A. Werk Cook professor of radiation oncology, appears in the October Nature Medicine. The researchers implanted a human gallbladder carcinoma into either the gallbladder wall or the subcutaneous space of the dorsal flank in mice and found that tumors grew faster in the gallbladder. In addition, plasma levels of TGF-beta1 were 300 percent higher in the group with cancer in the gallbladder. To learn whether a primary gallbladder tumor could suppress angiogenesis and tumor growth at a secondary site, the researchers then implanted tumors into the brains of the mice. Angiogenesis, tumor growth, and leukocyteendothelial cell interaction were inhibited in this secondary site in mice with gallbladder tumors. To establish whether TGF-beta1 was responsible for this suppression, the researchers then injected a neutralizing antibody to TGF-beta1 into the mice and found reversal of these inhibitory effects. TGF-beta1 also inhibited tumor cell growth in vitro. Gallbladder cancer is treated by surgical resection of the tumor. But this procedure cannot always prevent the rapid growth of distant metastases. The researchers believe that their findings, if confirmed in patients, "will have important implications for the treatment of gallbladder carcinoma" since modulation of TGF-beta1 levels after primary tumor resection might inhibit further spread of the cancer. Stem Cell Transplant Counters Muscular Dystrophy in MiceResearchers at HMS have made an important step toward gene therapy in people with muscular dystrophy. Severe forms of the disease are characterized by progressive muscle degeneration and early death due to a deficiency of dystrophin, a protein required for structural support of muscles. Previous attempts at gene therapy, although successful, have resulted in only local, and therefore temporary, dystrophin expression. Now HMS scientists report that they have been able to partially restore muscle dystrophin expression in mice by bone marrow transplantation of either hematopoietic stem cells or a novel population of muscle-derived stem cells. Both cell types were not only capable of incorporating dystrophin into muscle and expressing it, but were also able to reconstitute the bone marrow. These studies shed new light on the differentiation potential of various stem cell types. The findings by Richard Mulligan, the Mallinckrodt professor of genetics, and Louis Kunkel, HMS professor of pediatrics, both at Children's Hospital, and colleagues are reported in the Sept. 23 Nature. Addressing the Root Of the ProblemHighly purified hematopoietic stem cells derived from normal male mice were injected into lethally irradiated female mdx mice, an animal model of Duchenne's muscular dystrophy. After 12 weeks, the females' bone marrow was completely reconstituted with male donor cells, and donor cells were associated with dystrophin expression, which was found in up to 4 percent of muscle fibers. In separate experiments, the researchers isolated a unique population of cells from the skeletal muscle of normal male mice that displayed many stem cell characteristics. Bone marrow transplantation of these cells resulted in similar dystrophin expression and reconstitution of the bone marrow and spleen, although restoration was less efficient than with hematopoietic stem cells.Although the percentage of muscle fibers expressing dystrophin was low, the researchers believe the technique "could be optimized to provide levels of engraftment of muscle that would be clinically useful." They also believe this technique would be useful in treating other diseases in which systemic delivery of therapeutic cells would be beneficial. --This and briefs above by Lorene Leiter Peptide Found to Have Specific Action Against Immune ResponseResearch by Anjana Rao, HMS professor of pathology and senior investigator at the Center for Blood Research, and her colleagues suggests that a new era of immunosuppressive treatments might be just around the corner. Focusing their attention on the NFAT family of transcription factors, this team identified a peptide that can block the immune responses regulated by NFAT more specifically than drugs currently available for clinical use. (For background, see Focus May 1, 1998.) Upon activation by the calcium-regulated enzyme calcineurin, NFAT proteins migrate to the nucleus, where they promote the expression of genes responsible for effective immune responses. Contemporary immunosuppressants inhibit calcineurin at its active site, thereby interfering not only with NFAT activation but also with other metabolic functions of this phosphatase. These traditional drugs are known to have toxic side effects that prevent their use in treating chronic ailments in which NFAT has been implicated, such as allergy, arthritis, autoimmune diseases, and, potentially, myocardial hypertrophy. Rao's team collaborated with Michael Yaffe, HMS instructor in surgery, and Lewis Cantley, HMS professor of medicine and of cell biology, both at Beth Israel Deaconess, using a technique of iterative binding assays to develop the sequence of a peptide with a high affinity for the specific NFAT-binding domain of calcineurin. This peptide, named VIVIT for the amino-acid sequence in a critical portion of its binding region, attaches tightly to calcineurin and is more effective at inhibiting the interaction between these two proteins than peptides that mimic the natural calcineurin-docking sequences of NFAT. Tests confirmed that inhibition by the VIVIT peptide does not interfere with calcineurin's active site. The high specificity of the VIVIT peptide to inhibit the action of calcineurin on the NFAT transcription factor may offer an exciting alternative to the current treatments available. The high affinity of the VIVIT peptide to NFAT-binding domains also allows this compound to be used to identify NFAT target genes in various cell types. Potentially, the technique could be extended to block other signaling pathways by selectively interfering with specific proteinprotein interactions unique to each pathway. An article describing this team's work is published in the Sept. 24 Science, with postdoctoral fellow José Aramburu as first author. --Brief by Catherine Chu |
