John Nove

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Expression and Characterization of Bone Morphogenetic Protein-2 in Chinese Hamster Ovary Cells

Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.

The Repair of Potentially Lethal Damage in X-irradiated Cultures of Normal and Ataxia Telangiectasia Human Fibroblasts

The repair of potentially lethal damage (PLD) after X-rays was studied in plateau phase cultures of nine normal and five ataxia telangiectasia (AT) strains of human fibroblasts. In the normal strains PLD repair was complete after 6 hours of post-irradiation incubation. There were differences in the form of the survival curves of normal strains after maximum PLD repair but the extent of post-irradiation recovery was similar in all strains. In contrast all AT strains were almost completely deficient in PLD repair even when post-irradiation incubation was extended to 18 or 24 hours. The relevance of the PLD repair-deficiency in cultured AT strains to in vivo radiotherapeutic observations in AT patients is briefly discussed.

X-ray sensitivity of human tumor cells in vitro

Abstract Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined (n, D o ). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability.

Survival of human diploid skin fibroblasts from normal individuals after x-irradiation

The cytotoxic effect of X-rays was measured by a colony formation assay in multiple experiments with fibroblast cell strains derived from 24 presumably normal individuals, received as 65 different coded and blinded samples. Each strain was received on two or more occasions at different times and bearing different codes. The means and standard deviations of the survival curve parameters for the 24 strains were: D0 = 123 +/- 23; D10 = 273 +/- 42 cGy. The D0 ranged from 89 to 175 and the D10 from 196 to 372 cGy. The degree of interexperimental variation, though generally minimal, differed considerably among cell strains. There was no systematic effect of passage level, cloning efficiency, serum lot, age or sex of the donor on X-ray survival. These results confirm that the intrinsic radiosensitivity varies significantly among skin fibroblasts isolated from clinically normal individuals, apparently owing to as yet unidentified genetic factors.

Sensitivity of Human Diploid Fibroblast Cell Strains from Various Genetic Disorders to Acute and Protracted Radiation Exposure

The cytotoxic effect of acute X irradiation was studied by a colony formation assay in 114 human skin fibroblast cell strains from 31 apparently normal individuals and 83 patients with a variety of genetic disorders possibly associated with in vitro hypersensitivity to ionizing radiation. The effect of protracted exposure to beta radiation from tritiated water (HTO) was examined in parallel experiments in 65 of these strains. The disorders included neurological diseases and syndromes characterized by an increased susceptibility to spontaneous and radiation-induced cancer. Homozygous ataxia telangiectasia and Nijmegen break syndrome cells were highly sensitive to both types of radiation. However, the response of cells from the other genetic disorders fell within the broad range characteristic of normal cell strains. While HTO may be useful as a quantitative method for determining the cytotoxic response of human diploid cells to ionizing radiation, the present results indicate that it does not offer a more sensitive assay than acute X irradiation for detecting minor degrees of hypersensitivity.

Return of the chalones.

Members of the TGFbeta superfamily play many roles in embryonic development and adult tissue homeostasis. Now recent work focused on growth and differentiation factors (GDFs) suggest that these TGFbeta-like molecules may also control organ size and may, in fact, be the long sought after chalones, or negative growth regulators.

Retinoblastoma, chromosome 13, and in vitro cellular radiosensitivity

Five diploid fibroblast strains from patients with deletions mapping in the long arm of chromosome 13 and three strains bearing partial trisomies of chromosome 13 were assayed for clonogenic survival following X-irradiation. Results of these experiments suggest the existence of a region on 13q14 which is related to increased sensitivity to cell killing in vitro by X-rays. This locus seems to be distinct from but close to the retinoblastoma locus, which has been associated with the same band.

Interstitial deletion of chromosome 13 and associated congenital anomalies.

SummaryAn interstitial deletion of chromosome 13 with breakpoints at 13q22 and 13q32 is presented. The clinical findings associated with this deletion are discussed in relation to the correlations of specific chromosomal bands with constellations of congenital defects as described by Niebuhr and Ottosen (1973), Niebuhr (1977), Lewandowski and Yunis (1975), and Noel et al. (1976).