Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines.

OBJECTIVE: Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. MATERIALS AND METHODS: To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. CONCLUSION: The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo.

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

The production and characterization of murine monoclonal antibodies to human Gadd45 raised against a recombinant protein.

The production of two different murine monoclonal antibodies to human Gadd45, a protein that is induced in response to DNA damage, is reported. Antibodies were generated in a SJL mouse using a recombinant form of the human Gadd45 protein. Monoclonal antibody 4TCYA1, which recognizes the denatured form of human Gadd45 in Western blots, was selected based upon the recognition of Gadd45 induced by functional p53 in the human myeloid leukemia cell line, ML-1. A second monoclonal antibody, designated 30T.14, immunoprecipitates native human Gadd45 in lysates produced from RKO cells, a colorectal carcinoma cell line that expresses relatively high basal levels of Gadd45, as well as from cell lysates made from ML-1 cells after exposure to ionizing irradiation (IR). Since 4TCYA1 fails to immunoprecipitate Gadd45, and 30T.14 fails to bind to IR-induced Gadd45 in immunoblotting, these two monoclonal antibodies probably recognize different epitopes.

Alterations in the localization of F-actin, fibronectin, and thrombospondin occur prior to neoplastic transformation in rat tracheal epithelial cells.

Structural glycoproteins and cytoskeletal proteins play a major role in the regulation of cellular organization and function. Changes in the structure and function of these proteins are involved in the cascade of events which lead to neoplastic transformation. We evaluated RNA levels, protein localization, and organization of selected proteins in an in vitro model system of respiratory carcinogenesis to examine alterations in cell architecture. Localization of fibronectin (Fn), thrombospondin (Tsp), and F-actin was examined in (1) primary rat tracheal epithelial (RTE) cells; (2) spontaneously immortalized nonneoplastic cells (SPOC-1); and (3) neoplastic cells (EGV5T) derived from tumors arising following transplantation of an N-methyl-N'-nitro-N-nitrosoguanidine-transformed RTE cell line into nude mice. Proteins were stained with fluorescein-labeled antibodies or phalloidin compound and analysis was performed with a confocal laser scanning microscope. Primary RTE cells display organized F-actin stress fibers, perinuclear Fn and Tsp, and pericellular Fn in fibrillar arrays. In larger colonies, Tsp occurs between cells and occasionally in fibrillar arrays. SPOC-1 cells, unlike primary RTE cells and neoplastic EGV5T cells, seldom form junctions and exhibit few cell surface extensions. F-actin stress fibers are reduced in these immortalized cells. F-actin in SPOC-1 cells occurs in the perinuclear region, scattered diffusely throughout the cell and in punctate adhesions. Fn and Tsp are localized to the perinuclear region with Fn staining more intensely. EGV5T neoplastic cells also display a dramatic loss of stress fibers and F-actin is concentrated mainly near the cell periphery. Perinuclear staining of Fn and Tsp occurs in some cells within the colony. Levels of Tsp RNA and Fn RNA and protein are significantly reduced in both cell lines compared to primary RTE cells. We conclude that structural protein disruptions are early events in the transformation of these respiratory epithelial cells.

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.

Altered growth factor dependence and transforming growth factor gene expression in transformed rat tracheal epithelial cells.

The role of peptide growth factors in neoplastic progression of transformed rat tracheal epithelial (RTE) cells was assessed by examining growth factor requirements and expression of growth factor and growth factor receptor genes in normal and transformed RTE cells. Neoplastically transformed cell lines showed decreased requirements for bovine pituitary extract, insulin, and epidermal growth factor compared to normal primary RTE cells. Neoplastic RTE cell lines expressed significantly increased levels of transforming growth factor alpha (TGF alpha) RNA and secreted TGF alpha into the media, suggesting an autocrine role for this growth factor. Increased levels of TGF alpha RNA were also observed in the preneoplastic stages of the same cell lines, indicating that increased TGF alpha expression is an early event in the multistage process of neoplastic transformation of RTE cells. TGF beta transcripts were also overexpressed in neoplastically transformed cell lines. Our studies suggest that aberrant expression of growth factors may play an important role in the development and/or maintenance of the transformed phenotype in RTE cells.