Rose S. Fife

Inhibition of proliferation and induction of apoptosis by doxycycline in cultured human osteosarcoma cells

Matrix metalloproteinases (MMPs) play a major role in the phenomena of growth, invasion, and metastasis of malignant disease. We studied the effects of doxycycline, a synthetic tetracycline that has been shown to suppress MMP activity in other solid tumors, on osteosarcoma (OSA) cell proliferation and MMP activity in vitro. OSA cells from 6 patients and from one established human tumor cell line (U2OS) (American Type Culture Collection) were cultured in the presence or absence of doxycycline. Doxycycline (10 microg/ml) suppressed OSA cell proliferation threefold to sevenfold in all cultures. MMP activity was assessed by gelatin zymography and was diminished by approximately 50% in all cultures. We examined the hypothesis that induction of apoptosis is one of the mechanisms by which doxycycline inhibits OSA cell proliferation. Ethidium bromide-stained gels of DNA from cells grown in the presence of 5 microg/ml and 10 microg/ml of doxycycline revealed laddering consistent with apoptosis after 24 hours in culture. The demonstration that doxycycline suppresses cell proliferation and MMP activity and induces apoptosis in human OSA cells in vitro suggests that this well-tolerated oral agent may be effective in the in vivo treatment of OSA.

Effects of doxycycline on human prostate cancer cells in vitro

Prostate cancer is the most common form of cancer in older men and the major cause of death from prostate cancer is metastatic disease. The matrix metalloproteinases (MMPs) play a significant role in the growth, invasion and metastasis of many tumors, including those of the prostate. We previously demonstrated that doxycycline, a synthetic tetracycline, inhibits MMPs and cell proliferation and induces apoptosis in several cancer cell lines. We also demonstrated that in an in vivo model of metastatic breast cancer in athymic mice doxycycline inhibits tumor size and regrowth after resection. In the present study, gelatinolytic activity in the human prostate cancer cell line, LNCaP, was suppressed and significant inhibition of cell growth occurred after exposure to 5 or 10 microg/ml of doxycycline, while cell growth was normal in untreated cells. Radioisotope incorporation into proteins was reduced by doxycycline. DNA fragmentation, consistent with apoptosis, was demonstrated in cells treated with doxycycline. These data suggest that doxycycline may have potential utility in the management of prostate cancer.

Effects of vitamin D3 on proliferation of cancer cells in vitro

The principal cause of death from most forms of cancer is metastatic disease. Cancer cells appear to grow quickly out of the control of the normal host regulatory mechanisms. Many factors contribute to this unrestrained proliferation, including increased metalloproteinase activity causing degradation of the extracellular matrix surrounding cancer cells, angiogenesis permitting easy access of the cells to the bloodstream and decrease or loss of programmed cell death, or apoptosis, an important mechanism for removal of abnormal or senescent cells. Treatment modalities targeted towards arresting cancer cell proliferation and spread are needed to improve the survival of patients with cancer. Vitamin D3, 1,25-dihydroxychole-calciferol D3, has been shown to induce apoptosis in the human breast cancer cell line, MCF-7. We have studied the effects of three concentrations of vitamin D3 on the human breast cancer cell line, MDA-MB-435, the human prostate cancer cell line, LNCaP, and a human osteosarcoma cell line, U20S. We report here that vitamin D3 strikingly inhibits cell proliferation and induces apoptosis in all three cell lines.

Effects of tetracyclines on angiogenesis in vitro

Most tumors kill their hosts by the process of metastasis rather than by local growth of the primary mass. A significant factor contributing to the distant invasion of cancer cells is the ability of tumors to produce large numbers of new blood vessels in their midst, known as angiogenesis. This both provides access to nourishment for the primary cancer and enables the cells to escape from the tumor and enter the bloodstream. We have been examining agents that appear to inhibit metastasis and, in particular, angiogenesis. We now report on the ability of the synthetic tetracycline, doxycycline, and the chemically-modified tetracycline, COL-3, to inhibit angiogenesis in a quantitative in vitro assay of angiogenesis, using human umbilical vascular endothelial cells (HUVECs) attached to microcarrier beads.

Identification of a high-molecular-weight (> 400 000) protein in hyaline cartilage

Abstract A high-molecular-weight (> 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2–4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the > 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.

The origin of the intrinsic glycoproteins of the rabbit vitreous body : an immunohistochemical and autoradiographic study

A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1-22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic cross-reactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.

Vitreous contains a cartilage-related protein

The vitreous is a clear, gel-like mass in the posterior cavity of the eye. Its major components are type II collagen, hyaluronic acid, and water. A small amount of proteoglycan is also present. All of these molecules are found in cartilage as well. We have now identified a non-collagenous protein in bovine and human vitreous samples which has hitherto been found only in hyaline cartilage and fibrocartilage. This protein has a molecular weight of 550,000 and subunits of 130,000 MW and appears to be a shared cartilage and eye antigen. Its role, if any, in ocular disease associated with rheumatic disorders remains to be determined.

Identification of link proteins and a 116,000-Dalton matrix protein in canine meniscus

The menisci are collagen-rich, fibrocartilagenous structures which are important in protecting the articular cartilage of the knee from some of the impact of weight-bearing. Meniscal proteoglycans have been studied in several mammalian species, including the dog, but very little is known about the noncollagenous proteins of the menisci. In the present study, 4 M guanidinium chloride extracts of meniscal cartilage from normal adult mongrel dogs were studied, and several noncollagenous proteins, including the link proteins and a 116,000-Da subunit protein, which we have recently described in articular cartilage, were found in meniscal cartilage. The 116,000-Da subunit protein represents 3.8% of the total protein extracted from meniscal cartilage. The link proteins sedimented in the bottom of an associative cesium chloride density gradient, where high-buoyant-density proteoglycans sediment.

Cartilage matrix glycoprotein is present in serum in experimental canine osteoarthritis.

We have described previously a disulfide-bonded 550,000-D cartilage matrix glycoprotein (CMGP), which is found in normal hyaline cartilage, fibrocartilage, and the vitreous of the eye, and consists of subunits with apparent molecular weights of 130,000 in 4% gels (116,000 in 9% gels). In osteoarthritic cartilage from dogs subjected to transection of the anterior cruciate ligament (ACL), CMGP is cleaved to major immunoreactive fragments with apparent molecular weights of 65,000 and 75,000 after reduction with 2-mercaptoethanol. In the present study, using immunolocation analysis, a monoclonal antibody to CMGP did not react with serum from 8 of 12 dogs before ACL transection but did react with serum from seven of these animals 4 wk after surgery and with serum from 10 dogs at sacrifice, 8-14 wk after ACL transection. Serum from four dogs reacted with the monoclonal antibody before ACL transection. Serum from two dogs was negative at all time points. Immunolocation studies using a polyclonal antiserum to CMGP were performed in seven of these dogs and produced results identical with the monoclonal antibody in four dogs. In contrast, analysis of serial serum samples from three dogs with cartilage atrophy revealed no evidence of CMGP at any time point. These data suggest that CMGP may be a serum marker for osteoarthritis in this canine model.

Workgroup 2: Human Xenograft Models of Prostate Cancer

Mark E. Stearns (Chairperson),1* Joy L. Ware (Rapporteur),2 David B. Agus,3 Ching-Jer Chang,4 Isaiah J. Fidler,5 Rose S. Fife,6 Robin Goode,7 Eric Holmes,8 Michael S. Kinch,9 Donna M. Peehl,10 Thomas G. Pretlow II,11 and George N. Thalmann12 1Department of Pathology, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 2Department of Pathology, Virginia Commonwealth University, Richmond, Virginia 3Memorial Sloan-Kettering Cancer Center, New York, New York 4Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 5Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 6Indiana University School of Medicine, Indianapolis, Indiana 7Lilly Research Laboratories, Indianapolis, Indiana 8Pacific Northwest Cancer Foundation, Seattle, Washington 9Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 10Department of Urology, Stanford University, Stanford, California 11Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 12Universitat Bern, Bern, Switzerland