A comparative cell-based high throughput screening strategy for the discovery of selective tyrosine kinase inhibitors with anticancer activity.

Growth factor receptor tyrosine kinases (RTK) have been implicated in tumor growth, metastasis and angiogenesis, and are thus considered promising targets for therapeutic intervention in malignant diseases. We present a novel drug discovery strategy to find inhibitors of RTKs based on comparative screening of compound libraries employing functional cellular assays. Cell lines stably expressing HER2 and the receptors for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been established. All cell lines are based on FDC-P1, a murine myeloid progenitor cell line which allows a direct comparison of results obtained in primary screens. In addition, the same cell lines are suitable for compound optimization and for animal studies. Using this strategy we report the identification of promising lead candidates for further drug development which are highly selective, non-cytotoxic and cell permeable with potencies in the low micromolar range.

Generation of high-titer retroviral vectors following receptor-mediated, adenovirus-augmented transfection.

A new procedure is described for the generation of high-titer, helper-free retrovirus vectors employing receptor-mediated, adenovirus-augmented transfection into a standard packaging cell line. Viral titers are increased 30-fold to 100-fold in transiently (> 10(5) infectious units per mL) and stable (> 10(7) infectious units per mL) transfected cells as compared with either CaPO4-mediated transfection or retroviral infection of a packaging cell line. Further, expression of the transduced genes was drastically increased in the transfected cells, but, as expected, there was no difference in transduction efficiency and gene expression in the infected target cells. The increases in viral titers were most likely due to the high number of stable, integrated copies of the vector plasmid DNA in the resulting packaging lines following G418 selection. In addition, experiments generating recombinant retroviruses from non-packaging cell lines are presented. The results suggest that this procedure may be of use to generate high-titer retrovirus vectors in packaging cell lines as well as in primary cells, thus providing a technical basis for in vivo gene transfer upon transplantation of these cells into various organs.

Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal.

Overexpression of wild-type p53 in p53-deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild-type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin-3 (IL-3)-dependent cells. Extended survival without IL-3 was conferred by recombinant retroviruses encoding either a full-length p53 mutant or a C-terminal p53 miniprotein, both of which can act as negative-dominant inhibitors of wild-type p53. On the other hand, excess wild-type p53 activity failed to elicit apoptosis as long as IL-3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Rescue of W-associated mast cell defects in W/Wv bone marrow cells by ectopic expression of normal and mutant epidermal growth factor receptors.

The normal human epidermal growth factor receptor (EGF-R) (HERc), a chimeric EGF-R/v-erbB (HERerbB) receptor, and the ligand-independent oncogenic EGF-R variant (v-erbB) were used to correct the mast cell defects in W/Wv bone marrow (BM) cells. In culture, all three receptor molecules transduced functional mitogenic signals in infected interleukin-3 (IL-3)-dependent bone marrow-derived mast cells (BMMCs) and enabled their differentiation into safranin-positive mast cells resembling connective tissue-type mast cells (CTMCs). Furthermore, expression of these receptors restored the capacity of W/Wv BMMCs to colonize the peritoneal cavity of mast cell-deficient W/Wv mice where they differentiated to safranin-positive cells with similar frequencies as wild-type BMMCs. These experiments show that expression of normal and mutant EGF-Rs in W/Wv BM cells is able to complement the function of the c-kit-encoded Steel factor receptor (SLF-R) in mast cell development. We conclude that signal transduction by normal and mutant EGF-Rs in murine hematopoietic cells apparently involves components also used by the SLF-R, which suggests that these receptors use overlapping pathways for signal transduction.

Development of a lethal mast cell disease in mice reconstituted with bone marrow cells expressing the v-erbB oncogene.

An animal model for malignant mastocytosis is described in mice reconstituted with bone marrow cells expressing the v-erbB oncogene. The lethal mast cell disease is characterized by massive infiltration of bone marrow, spleen, and several other visceral organs by connective tissue mast cells, which normally reside in the skin and the peritoneal cavity. As is frequently found in malignant mastocytosis, the v-erbB-induced mast cell disease was accompanied in some primary recipients by an acute myelogenous leukemia (AML) that killed all secondary recipients regardless of whether the AML was already evident in the primary host. The infiltrating mast cells stained strongly positive with berberine sulfate, suggesting that they were terminally differentiated and in vitro they showed only a weak proliferative capacity. The leukemias were clonal but apparently of different origin than the malignant mast cells, implying the transformation of two independent cell populations. Leukemic cells expressed various myeloid-specific markers as well as the B220 antigen, normally associated with the B-cell lineage. However, the Ig heavy chain genes were still in germ line configuration. In culture, these cells proliferated in the absence of exogenous growth factors and had the capacity to differentiate into mature myeloid cells. Preliminary experiments suggest that v-erbB may use parts of a signal transduction pathway normally coupled to the c-kit receptor. The v-erbB-induced malignant mast cell disease should provide a useful animal model for elucidating the cause for malignant mastocytosis in humans and to explore possible therapeutic strategies.

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.

Insulin-like growth factor-1 stimulates proliferation of myeloid FDC-P1 cells overexpressing the human colony-stimulating factor-1 receptor.

Retrovirally expressed human CSF-1 receptor can induce CSF-1-dependent growth of IL-3-dependent hemopoietic cells FDC-P1. Here we show that expression of the human CSF-1 receptor also allowed FDC-P1 cells to grow in response to Insulin-like Growth Factor-1 (IGF-I). The authentic receptor for IGF-I was identified by affinity cross-linking and binding analysis on both control (infected with a neo vector) and CSF-1 receptor expressing FDC-P1 cells. DNA and RNA analysis of these cells and of five clones of IGF-I responsive cells demonstrated that the IGF-I receptor gene was not rearranged nor was it abnormally expressed in IGF-I responsive cells. These results suggest that myeloid cells over-expressing CSF-1R (c-fms protooncogene product) might have a proliferative advantage over normal myeloid cells in a physiological situation, independently of the presence of CSF-1 or the capacity of the cells to respond to CSF-1. This would indicate a possible role for c-fms in human neoplasia.

Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.

In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi- and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro.

Expression of human CSF-1 receptor induces CSF-1-dependent proliferation in murine myeloid but not in T-lymphoid cells.

The receptor for human macrophage colony stimulating factor (CSF-1R) was introduced into hematopoietic cell lines of myeloid and T-lymphoid origin, both of which normally do not express the CSF-1R. Infection of an interleukin-3 (IL-3)-dependent mouse myeloid cell line (FDC-P1) with a high titer retroviral vector expressing the human c-fms c-DNA, enabled CSF-1-dependent proliferation in short-term liquid culture assays as well as in clonal culture systems. CSF-1-dependent cell lines could be established after sorting for CSF-1R positive cells. In contrast to FDC-P1 cells, expression of the CSF-1R in CTLL cells, an IL-2-dependent mouse cytotoxic T-cell line, and in T-cell growth factor III/P40-dependent helper T-cells, ST2/K9.4a2, did not lead to CSF-1-dependent proliferation. These observations lead to the conclusion that ectopically expressed CSF-1R may function on certain myeloid cells where it is normally not expressed, suggesting the presence of signal transduction pathways which can be utilized by that foreign receptor. In contrast, it appears that T-lymphoid cells lack such a signalling mechanism, indicating that quite different modes of transducing mitogenic signals from the cell membrane to the nucleus must have developed during myeloid and T-lymphoid differentiation.

Inhibition of human T-cell leukemia virus type I replication in primary human T cells that express antisense RNA.

The human T-cell leukemia virus type I is associated with adult T-cell leukemia-lymphoma in humans, a disease which is induced by a malignant transformation of T lymphocytes. Retrovirus vectors carrying human T-cell leukemia virus type I-derived sequences in reversed transcriptional orientation were used to express antisense RNA transcripts in primary human leukocytes. Human T-cell leukemia virus type I replication and virus-mediated immortalization were inhibited in cells harboring antisense constructs. This study suggests that retrovirus-mediated antisense RNA inhibition can be used to protect primary human T-lymphocytes from human T-cell leukemia virus type I-mediated cell transformation.

Expression of functional human EGF receptor on murine bone marrow cells.

The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines.

Effect of internal viral sequences on the utility of retroviral vectors.

Expression of the human ADA cDNA encoded by the Moloney murine leukemia virus spliced RNA form is enhanced by intron-contained sequences. The presence of sequences corresponding to the viral gag gene in a Moloney murine leukemia virus-based vector results in the generation of 10- to 40-fold higher titers of virus.

Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.

A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.