Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models.

During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC(50) of 110 and 1980 nM, respectively, and has good pharmacokinetic profile in mouse and dog. We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED(50)) decreased with increasing levels of VEGF (r=-0.94); and, in contrast, the ED(50) increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Misregulated expression of the cyclin dependent kinase 2 protein in human fibroblasts is accompanied by the inability to maintain a G2 arrest following DNA damage.

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.

Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1 beta. Thus, TCDD alters the expression of growth regulatory genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture.

A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells.

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin and thyroid hormones act through different mechanisms in human keratinocytes.

It has been proposed [J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41-53] that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361-1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T3) and T4 on human target cells. Keratinocytes were treated simultaneously with T3 and T4 in a 4:1 molar ratio (T3/T4; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P1-450 induction) were measured. T3/T4 (at concentrations up to 10 microM T3/2.5 microM T4) and T3 or T4 alone (0.1 to 10 microM) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T3/T4 was not due to metabolism of these hormones by the keratinocytes. T3 and T4 did not compete with [3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [125I]T3 or [125I]T4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.

An in vitro model for studying the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to human thymus.

A coculture system of human thymic epithelial (HuTE) cells and thymocytes (T lymphocyte precursors) has been established and characterized as an in vitro model for assessing the potential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to human thymus. HuTE cells in culture were responsive to TCDD as judged by induction of the cytochrome P1-450 monooxygenase activities, 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD). Measurement of the responsiveness of thymocytes cocultured on TCDD-pretreated HuTE monolayers to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) indicated that TCDD can act directly on HuTE cells to suppress thymocyte maturation (at a concentration of 10 nM, TCDD produced a 25 to 50% inhibition of thymocyte responsiveness to Con A and PHA). Both the induction of cytochrome P1-450 monooxygenase activity (EC50 values approximately 1 nM) and immunosuppressive responses elicited by TCDD in HuTE cells were concentration-dependent and stereospecific (as judged by the relative activities of chlorinated dibenzo-p-dioxin and dibenzofuran isomers), indicating involvement of the Ah receptor which was detected in all HuTE strains examined. Initial characterization of these Ah receptor-mediated responses in several strains of HuTE cells indicated marked interstrain differences in maximally inducible ECOD and EROD activities which did not appear to directly correlate with measured concentrations of the cytosolic Ah receptor, and in certain strains examined, differences in sensitivity and magnitude were observed for TCDD-evoked immunotoxic responses but not always for the induction response. These data on the actions of TCDD on cultured HuTE cells suggest that human thymus is a target for TCDD and related halogenated aromatic compounds. In HuTE cells, measurement of either the Ah receptor concentration or of marker responses such as the induction of cytochrome P1-450 alone cannot provide an accurate quantitative assessment of susceptibility to TCDD-induced thymus toxicity.

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture.

In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis, and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes. Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration dependence and stereospecificity for these responses suggest the involvement of the Ah receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating basal cells, modulating the responsiveness of these cells to growth factors such as EGF.

Evidence for direct action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL/6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 nM) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 nM TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 nM) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC50 approximately 1 nM) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.

Toxicity of chlorinated aromatic compounds in animals and humans: in vitro approaches to toxic mechanisms and risk assessment.

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and chlorinated analogs commonly results in pathological changes in the skin and its appendages characterized by thickening of the epidermis (acanthosis), hyperkeratosis and squamous metaplasia of the epithelial lining of the sebaceous glands. Acneform lesions (chloracne) develop as hair follicles dilate and fill with keratin and sebaceous glands become cystic. In animal models it has been found that the chloracneogenic potential of the halogenated aromatic compounds examined corresponds with the relative affinity of these same compounds for the cytosolic TCDD receptor. This receptor controls the coordinate expression of a number of inducible enzyme activities and in certain cell targets can alter normal programs of proliferation and differentiation. In this report we describe some of our ongoing studies on the mechanisms of action of TCDD in normal human epidermal cells and squamous cell carcinoma (SCC) lines. These systems permit detailed investigation of the molecular and biochemical events underlying pathologic changes in the skin and offer the potential of establishing a risk assessment model for halogenated aromatic compounds by using human target cells.