Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma.

In a transgenic mouse model, dermal fibrosarcomas develop in a pathway comprised of at least three stages: mild fibromatosis, aggressive fibromatosis, and fibrosarcoma. The latter two stages are highly vascularized when compared with both the normal dermis and the initial mild lesion. Analysis of cell cultures derived from biopsies of these lesions has revealed that basic fibroblast growth factor (bFGF) is synthesized in all three stages and in normal dermal fibroblasts derived from the same mice. Unexpectedly, there is a change in the localization of bFGF from its normal cell-associated state to extracellular release in the latter two stages, which is concomitant both with the neovascularization seen in vivo and with the tumorigenicity of these cell lines. Thus, in this multistep tumorigenesis pathway there appears to be a discrete switch to the angiogenic phenotype that correlates with the export of bFGF, a known angiogenic factor.     

Loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development

Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages. Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown. Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression. Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions. This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism. Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC. These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development. To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.     

Loss of Mitotic Spindle Checkpoint Activity Predisposes to Chromosomal Instability at Early Stages of Fibrosarcoma Development

Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages. Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown. Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression. Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions. This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism. Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC. These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development. To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.     

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.     

Loss of glucocorticoid receptor expression by DNA methylation prevents glucocorticoid induced apoptosis in human small cell lung cancer cells

Human small cell lung cancer (SCLC) is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR) expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.     

Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts

Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

Comparison of exogenous growth stimuli for chemically transformed cells: growth factors, serum and cocultures

Rat tumor cells isolated from 2 fibrosarcomas which differed in their degree of differentiation were exposed to growth-stimulating influences in soft agar. The effects of growth factors (epidermal growth factor, fibrosarcoma growth factor and insulin), of fetal calf serum and of cocultured normal mesenchymal cells of rats and nude mice were compared. Each of the 3 growth factors exerted a specific response and dose dependence. Increasing concentrations of serum stimulated the number of cells which formed clones in soft agar. Experiments using combinations of growth factors and fetal calf serum demonstrated that a complex optimal mixture of growth stimuli was responsible for the efficient growth-promoting activity of the serum. In cocultures with normal cells, cloning efficiency of tumor cells was enhanced and growth of tumor cells was accelerated. This stimulus was due to the constant release of an agar-diffusible growth-stimulating factor by the normal, nondividing cells. Cocultured mouse cells showed an even higher growth-stimulating activity than rat fibroblasts. Cells obtained from the poorly differentiated fibrosarcoma responded, in relative terms, better to all growth-stimulating influences, than those derived from the well-differentiated tumor.