Zebrafish developmental screening of the ToxCast™ Phase I chemical library.

Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concentration range of 1 nM-80 µM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chemicals were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chemical class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.

The effects of orientation, temperature, and displacement magnitude changes on the sonometrics system accuracy.

The accuracy and reliability of a sonomicrometry system (Sonometrics Corporation, Ontario, Canada) was evaluated for its potential use in measuring 3-D in vivo joint kinematics. Distances between different sets of piezoelectric crystals were measured through a salt solution using ultrasound technology. We evaluated crystal-to-crystal distance under simulated in vivo conditions of changing crystal orientation and displacement magnitude. Crystal-to-crystal distance was also evaluated under changing solution temperature, since the crystals may be used at different temperatures. The 2 mm round and peg crystals were accurate to within 0.5mm for 0 through 180 degrees rotations, but the 2mm round suture loop crystals were only reliable at 0 degrees rotation. The speed of sound through a salt solution (and hence the distance between crystals) versus temperature was fit using a second order polynomial, C=1421.1+3.9808T-3.09x10(-2)T2, with an R2 value of 0.9998. The translational error was less than 0.072 mm for crystal displacements of 0.012, 0.2, 1.0, and 5.0 mm. The system was also accurate under dynamic conditions with translational errors that were less than 0.045 mm under 0.65 Hz motion. These results suggest that the Sonometrics crystals possess attributes (translational accuracy and rotational independence) that could provide the basis for a system capable of measuring joint kinematics.

Manipulation of cellular interactions with biomaterials toward a therapeutic outcome: a perspective.

Manipulation of the wound healing process and the manner in which tissues interact with inert biomaterials were both made possible with the discovery of arginine-glycine-aspartic (RGD) acid as a major cell recognition signal in the extracellular matrix. Whether promoting cell adhesion or selectively inhibiting cell-cell aggregation mediated by integrin cell surface receptors, RGD-containing peptides can be rationally designed to incorporate both stability and integrin specificity. Synthetic peptides containing this sequence have been linked to biodegradable biopolymers and introduced for the enhancement of dermal and corneal would healing. By accelerating the healing reaction using RGD-containing peptides, the quality of regenerated tissue seems to be improved, the extent of fibrosis restricted, and the risk of microbial infection may be reduced. Controlling the degree of fibrosis that often accompanies the healing of wounds and the reaction of tissue to foreign materials can also be achieved by natural antagonists of fibrogenic activity of TGF-beta animal models of kidney fibrosis. These advances in the biotechnology of wound healing and tissue regeneration eventually will have an overall impact on the quality of health care.

Altered regulation of protein disulfide isomerase in cells resistant to the growth-inhibitory effects of transforming growth factor beta 1.

A murine keratinocyte cell line that is resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF beta 1) was examined for differential gene expression patterns that may be related to the mechanism of the loss of TGF beta 1 responsiveness. Cells that were resistant to the growth-inhibitory effects of TGF beta 1 (KCR cells) were derived from K-ras-transformed BALB/MK keratinocytes (KC cells). Using a subtractive hybridization procedure with KC and KCR mRNAs, we isolated a complementary DNA clone for murine protein disulfide isomerase (PDI). The mRNA for PDI is inhibited by TGF beta 1 treatment in the parental KC cells, but not in the TGF beta 1-resistant KCR cells. Similar PDI down-regulation also occurs in other TGF beta-sensitive cells, but not in a human pancreatic carcinoma cell line which is insensitive to the growth-inhibitory effects of TGF beta 1. The results suggest that misregulation of PDI, an important component of co- and posttranslational modification systems, may be involved in the mechanism by which some cells escape from the growth-inhibitory effects of TGF beta 1.

Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta.

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.

Transforming growth factor beta in the control of epidermal proliferation.

Transforming growth factor beta is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF beta in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF beta and TGF alpha undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.

Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.

Transforming growth factor beta (TGF beta) is a potent inhibitor of epithelial cell proliferation. A nontumorigenic epidermal growth factor (EGF)-dependent epithelial cell line, BALB/MK, is reversibly growth arrested by TGF beta. TGF beta will also abrogate EGF-stimulated mitogenesis of quiescent BALB/MK cells. Increased levels of calcium (greater than 1.0 mM) will induce differentiation in BALB/MK cells; in contrast, TGF beta-mediated growth inhibition does not result in induction of terminal differentiation. In the present study, the effects of TGF beta and calcium on growth factor-inducible gene expression were examined. TGF beta markedly decreased c-myc and KC gene expression in rapidly growing BALB/MK cells and reduced the EGF induction of c-myc and KC in a quiescent population of cells. TGF beta exerted its control over c-myc expression at a posttranscriptional level, and this inhibitory effect was dependent on protein synthesis. TGF beta had no effect on c-fos gene expression, whereas 1.5 mM calcium attenuated EGF-induced c-fos expression in quiescent cells. Expression of beta-actin, however, was slightly increased in both rapidly growing and EGF-restimulated quiescent BALB/MK cells treated with TGF beta. Thus, in this system, TGF beta selectively reduced expression of certain genes associated with cell proliferation (c-myc and KC), and at least part of the TGF beta effect was at a posttranscriptional level.

Growth modulation of mouse keratinocytes by transforming growth factors.

The effects of exogenously added transforming growth factor (TGF alpha and TGF beta on the growth of BALB/MK cells were examined. TGF alpha supplanted the epidermal growth factor (EGF) requirement in these cells. In contrast, TGF beta reversibly inhibited the growth of BALB/MK cells by abrogating the stimulatory actions of EGF or TGF alpha. The inhibitory effects of TGF beta appeared to be mediated by events distal to EGF ligand-receptor interactions. Growth inhibition of BALB/MK cells by TGF beta did not result in the induction of differentiation. This finding is different from the growth inhibition of these cells induced by elevated calcium levels (1.5 mM) which was tightly coupled to terminal differentiation. The BALB/MK cells were found to express TGF alpha mRNA, as well as TGF beta mRNA and protein. In addition, TGF alpha, as well as EGF, enhanced TGF alpha gene expression. These studies suggest a role for endogenous TGFs in regulating BALB/MK proliferation. TGF alpha provides a positive growth signal, while TGF beta is a potent inhibitor of growth even in the presence of such positive modulators as TGF alpha and EGF.

Transformation-related growth factors and their receptors.

Cellular transformation may be accomplished in vitro and in vivo through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in growth factor-signal transduction pathways that normally operate to control proliferation. Activation of genes that code for growth factors and/or their receptors provides tumor cells with potential mechanisms to maintain their proliferative state. Tumor cells have been shown to produce endogenous substances that augment their growth (autocrine stimulation), as well as responding to exogenous substances (paracrine stimulation). With solid tumor cells these responses have been shown to involve aberrant expression of growth factor and/or receptor genes. The study of the interrelationship of these various growth regulatory molecules is important not only in the identification of gene products essential to cellular proliferation, but also in providing clues as to what forces are driving tumor cell growth.

Biological characterization of an acid-sensitive growth factor from human platelets: role in the proliferation of human melanoma and bovine endothelial cells.

An acid-sensitive growth factor for human melanoma has been partially purified from human platelets. TSK gel filtration HPLC provides a molecular weight estimation of 60,000 daltons. This factor is not only mitogenic for human melanoma, but also stimulates 3H-thymidine uptake and increases the number of bovine aortic endothelial cells, while fibroblasts are nonresponsive. Radioiodination of active HPLC fraction has been accomplished. The human melanoma cell line, M1RW5 demonstrates specific, time-dependent binding of the labeled protein. These studies suggest that the growth of human melanoma may in part be regulated by a newly described growth factor present in human platelets.

Stimulation of human metastatic melanoma colony-forming cells by an acid-sensitive factor in human platelet sonicate.

A human platelet sonicate was evaluated for its effects on the growth of human metastatic melanoma colony-forming cells in soft agar from cells in culture and from biopsies. The addition of platelet sonicate increased both cloning efficiency and proliferative capacity in that more and larger colonies were formed. In more detailed studies under growth-limiting conditions, melanoma cellular responses to known growth factors were compared to the activity found in the platelet sonicate. None of the growth factors tested either alone or in combination, including platelet-derived growth factor, epidermal growth factor, alpha-type transforming growth factor, and beta-type transforming growth factor, were capable of inducing melanoma colony formation to the 12-fold stimulation observed with the platelet sonicate. Treatment of platelet sonicate with dithiothreitol, trypsin, or acid resulted in loss of activity for human melanoma. Our results suggest that human platelets contain an acid-sensitive protein which can support the expression of the transformed phenotype of human melanoma, and this factor is distinct from acid-stable activities previously characterized from human platelets.