Paul C. Billings

Heparan Sulfate Proteoglycans (HSPGs) Modulate BMP2 Osteogenic Bioactivity in C2C12 Cells

Cell surface heparan sulfate proteoglycans (HSPGs) have been implicated in bone morphogenetic protein (BMP)-mediated morphogenesis by regulating BMP activity and gradient formation. However, the direct role of HSPGs in BMP signaling is poorly understood. Here we show that HSPGs directly regulate BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts. HSPGs sequester BMP2 at the cell surface and mediate BMP2 internalization. Depletion of cell surface HSPGs by heparinase III treatment or decreased glycosaminoglycan chain sulfation with sodium chlorate enhances BMP2 morpho-genetic bioactivity. The addition of exogenous heparin, a widely used anticoagulant, reduced BMP2 signaling. Our results suggest that cell surface HSPGs mediate BMP2 internalization and modulate BMP2 osteogenic activity.

The TGFβ-inducible matrix protein βig-h3 interacts with fibronectin

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, βig-h3, is a highly conserved transforming growth factor-β-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length βig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on βig-h3-coated plates and developed actin stress fibers. Purified βig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the βig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of βig-h3 in the ECM.

Protease Activity in a Hapten-Induced Model of Ulcerative Colitis in Rats

Inflammatory bowel disease (IBD) is a painfuland debilitating condition affecting the mucosal liningof the colon and other areas of the gastrointestinaltract. IBD generally falls into two major categories: ulcerative colitis (UC) and Crohns disease. Wehave utilized dinitrobenzenesulfonic acid (DNBS) toinduce experimental UC in rats. Histopathologic analysisindicates that DNBS induces a condition in animals similar to human UC. Biochemical resultsrevealed 6- to 10-fold elevated levels of serineprotease activity in colon tissue from animals with UCas compared with matched controls. We also observedelevated levels of protease activity in tissue samplesobtained from human patients with UC. Hence, our resultsdemonstrate that protease activity is increased inrodent and human UC. These proteases may play a significant role in destruction of colonictissue in IBD. Protease inhibitors that target serineproteases may be useful pharmacological agents to limittissue destruction in IBD.

Effects of Bowman-Birk inhibitor on rat colon carcinogenesis.

The present study was undertaken to determine whether the Bowman-Birk inhibitor (BBI) could prevent colon carcinogenesis in rats treated with dimethylhydrazine (DMH) and whether there were adverse side effects associated with treatment with BBI for cancer prevention. BBI was evaluated in the forms of purified BBI (PBBI) or an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). The results demonstrate that PBBI and BBIC reduced the incidence and frequency of tumors in DMH-treated rats compared with animals treated with DMH alone. Autoclaved BBIC, in which the protease inhibitor activity of BBI was destroyed, had a weak and statistically insignificant, suppressive effect on DMH-induced colon carcinogenesis in rats, suggesting that the protease inhibitor activity of BBI is likely to be responsible for the anticarcinogenic activity of BBIC. Soy molasses, which contains soy isoflavones, did not have an effect on colon cancer carcinogenesis in DMH-treated rats. Similar to results from previous studies (Nauss et al. JNCI 73, 915-924, 1984), the most aggressive, malignant colon adenocarcinomas developed within or in association with gut-associated lymphoid tissue aggregates. No adverse side effects on the pancreas or animal growth were observed in rats treated with PBBI or BBIC. These results demonstrate that PBBI and BBIC may be used to prevent colon cancer without significant adverse side effects.

ANTICARCINOGENIC ACTIONS OF PROTEASE INHIBITORS

There is a great variation in cancer incidence with diet, as has been recently reviewed (1,2). Epidemiological data suggest that environmental, specifically nutritional, factors play a major role in the etiology of cancer at many different sites (1–3). There are now several epidemiologic studies which suggest that components of vegetables might play a beneficial role in lowering the incidence of cancer (some examples of such studies are given in references 1-4). Although many compounds with anticarcinogenic potential are present in vegetables, it is possible that anticarcinogenic protease inhibitors contribute to the low cancer rates observed in certain human populations with high levels of vegetables in the diet. For example, the low cancer incidence rates in the Japanese and Seventh-Day adventists could be due to high levels of dietary protease inhibitors; it has been estimated that individuals in these populations ingest, on the average, more than 330 mg of protease inhibitors per day (3). There are, however, many other hypotheses which have been presented to explain the low cancer rates in these human populations. So many different variables are present in the diet that the effect of any specific anticarcinogenic agent cannot be distinguished in such epidemiologic studies; however, it is possible to distinguish the effects of specific anticarcinogenic agents in laboratory experiments. In this report, our own laboratory studies on anticarcinogenic protease inhibitors will be summarized and discussed. Although laboratory studies can give much information about the effects of potential chemopreventive agents, ultimately epidemiologic intervention studies must be performed to determine whether candidate chemopreventive agents are truly capable of preventing cancer in human populations. The current evidence that dietary protease inhibitors do have a role in lowering the cancer incidence in human populations has recently been reviewed (6 and 7).

C-Myc expression is reduced in antipain-treated proliferating C3H 10T12 cells

In this report, we demonstrate that treatment of proliferating irradiated and nonirradiated C3H 10T1/2 cells with the protease inhibitor antipain is associated with a reduction in c-myc expression. Under conditions in which antipain treatment results in reduced c-myc transcripts, there is no effect on total RNA synthesis, growth rate, or saturation density. Antipain may be a useful inhibitor in which to further study the role of c-myc in cellular physiology.

Genetic testing in the workplace: a view from the USA

Progress in human genetics, accelerated by the Human Genome Project, is leading to a rapid proliferation in the number of genetic tests. Although they have their benefits, genetic tests have in the past been used by various societal institutions, including employers, to discriminate against and stigmatize individuals. In the light of the increase in the number of tests and some current reports of discrimination in employment, a variety of legal and regulatory measures have been proposed to prevent genetic discrimination in the workplace.

Expression and methylation of the Blym gene in human tumor cell lines

We have examined Blym expression in 11 human tumor cell lines. Increased Blym expression was observed in one of three osteosarcoma cell lines relative to nontransformed human foreskin fibroblasts. In addition, enhanced Blym expression was observed in a melanoma cell line and in 2 of 6 squamous carcinoma cell lines relative to nontransformed, low passage human epithelial cells. We found no evidence of gene amplification or rearrangements of Blym sequences in any of the cell lines we have examined. We further analyzed the state of methylation of the Blym gene in several of the tumor cell lines by Msp I/Hpa 11 restriction endonuclease digestion. All cell lines examined had similar Msp I digestion patterns. However, the different tumor cell lines had different Hpa 11 digestion patterns. Therefore, our results indicate that the Blym gene is differentially expressed and methylated in human tumor cell lines.