Ha-ras Oncogene Research Articles

Simvastatin Inhibits Malignant Transformation Following Expression of the Ha-ras Oncogene in NIH 3T3 Fibroblasts

In previous studies we have shown that the expression of the transformingHa-ras oncogene in NIH 3T3 fibroblasts stimulates cellular calcium entry, which triggers oscillatory calcium induced calcium release from internal stores. The intracellular calcium oscillations lead to cytoskeletal remodeling by actin stress fiber depolymerization and activation of the Na⁺/H⁺ exchanger thus mediating cell swelling and intracellular alkalosis, both important mitogenic signals. This is evidenced by abrogation of Ha-ras induced growth factor independent cell proliferation by interference with any of these events, i.e. by inhibition of cellular calcium entry or inhibition of the Na⁺/H⁺ exchanger.As shown in this study, simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme for cholesterol biosynthesis, is able to prevent these events following the expression of the transforming Ha-ras oncogene. We show, that simvastatin inhibits farnesylation dependent membrane translocation of a CAAX motive bearing yellow fluorescent protein and suppresses Ha-ras stimulated cellular calcium influx, which can be identified as capacitative calcium entry. In addition simvastatin is able to block regulatory volume decrease channels and to suppress the cytoskeletal remodeling, intracellular alkalinization, increase in cell volume and growth factor independent cell proliferation induced by the oncogene.Thus simvastatin is able to prevent crucial cellular events following expression of the transforming Ha-ras oncogene.

Mutation of Ha-ras Oncogene in Rat Salivary Gland Tumors Induced by DMBA.

The incidence of salivary gland tumor is approximately 2% among all head and neck tumors, of which malignant cases account for only about 5%. Much research has been performed in order to clarify the mechanism of oncogene activation, however salivary gland tumors remain understudied. We performed this study in order to characterize the ras gene in these tumors. We treated white rats with 7, 12-dimethylbenz[a]anthracene (DMBA) and confirmed the occurrence of salivary gland tumors after ten to thirty weeks. Isolated genomic DNAs from tumor tissues were added to NIH 3T3 cells. In order to detect Ha-ras mutations, we performed a two-step PCR-RFLP and 7analyzed the mutated sequences. We induced salivary gland tumors by DMBA treatment in white rats. Isolated DNAs from the tumor tissues transformed the NIH 3T3 cells. Point mutations were observed in codons 12 and 61 of the Ha-ras oncogene. The total frequency of point mutations was 13.9% in DMBA-induced salivary gland tumors in rats. Our results demonstrate that a variety of cancers ras oncogene mutations were also found in salivary gland tumors. We confirmed that a point mutation of the Ha-ras oncogene in a DMBA-induced salivary gland tumor occurs at a frequency of 13.9%.

Inhibition of cell transformation by sulindac sulfide is confined to specific oncogenic pathways

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce the risk of colorectal cancer (CRC). They are also known to induce the regression of colorectal adenomas, which are precursors to CRC. Despite these evidences, the exact mechanism by which NSAIDs exerts its anti-oncogenic effect is not completely understood. Using a focus formation assay, here we show that sulindac sulfide, a NSAID, specifically inhibits cell transformation mediated by oncogenic Ha-Ras, but not by other established oncogene products such as v-Src, G α12, and G α13. Our results suggest that the ability of sulindac sulfide to suppress transformation is confined to specific oncogenic pathways. Further studies of the sulindac-resistant oncogenic pathways may lead to identification of novel therapeutic agents that are effective in the prevention or treatment of CRC.

A Direct Interaction between Oncogenic Ha-Ras and Phosphatidylinositol 3-Kinase Is Not Required for Ha-Ras-dependent Transformation of Epithelial Cells

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.

Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras.

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEK- and Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating that aPKC-zeta functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon and zeta act downstream of Rac and are required for the activation of ERKs.

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42.

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.

Occupational cancer genetics: infrequent ras oncogenes point mutations in lung cancer samples from chromate workers.

Chromium carcinogenicity and mutagenicity are no longer disputed. However, although chromium has various genetic effects that induce cancer, its mechanism of inducing lung cancer in humans is still not fully understood. p53, a tumor suppressor gene, was found to be infrequently mutated in samples of lung cancer in workers with long occupational exposure to chromium, suggesting other cancer-related genes to be targeted in such tumors. To assess the contribution of the ras oncogenes in the pathogenesis of chromate-related lung cancer, we studied point mutations at the critical positions of codons 12, 13, and 61 of the Ha-ras and Ki-ras oncogenes in 38 lung cancer samples derived from Japanese patients who worked in the chromate industry for long periods. We used both radioactive isotope and non-radioisotope PCR-SSCP techniques. The results of this study demonstrated that activation of ras genes due to point mutations in chromate-related lung cancer is a rare event. Ras oncogenes activated by point mutations do not have a major role in the process of tumorigenesis of chromate-related lung cancer.

Akt/PKB Activity Is Required for Ha-Ras-mediated Transformation of Intestinal Epithelial Cells

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity.

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

Intestinal-enriched Krüppel-like Factor (Krüppel-like Factor 5) Is a Positive Regulator of Cellular Proliferation

Intestinal-enriched Krüppel-like factor (IKLF or KLF5) belongs to the family of mammalian Krüppel-like transcription factors. Previous studies indicate that expression of IKLF is enriched in the proliferating crypt epithelial cells of the intestinal tract. However, the biological function of IKLF is unknown. In the current study, we have shown that the level of IKLF mRNA was nearly undetectable in serum-deprived NIH3T3 fibroblasts but became acutely and significantly increased upon the addition of fetal bovine serum or the phorbol ester, PMA. This induction required protein synthesis because it was prevented by cycloheximide. Transfection of IKLF into NIH3T3 cells resulted in the formation of foci in a manner similar to that caused by the activated Ha-ras oncogene. Constitutive expression of IKLF in transfected NIH3T3 cells significantly increased the rate of proliferation when compared with cells transfected with an empty vector. The growth of IKLF-transfected cells was no longer inhibited by cell-cell contact or by low serum content. Moreover, these cells proliferated in an anchorage-independent fashion. We conclude that IKLF encodes a delayed early response gene product that positively regulates cellular proliferation and may give rise to a transformed phenotype when overexpressed.

Role of the Cytosolic Phospholipase A2-linked Cascade in Signaling by an Oncogenic, Constitutively Active Ha-Ras Isoform

Activation of Ras signaling by growth factors has been associated with gene regulation and cell proliferation. Here we characterize the contributory role of cytosolic phospholipase A(2) in the oncogenic Ha-Ras(V12) signaling pathway leading to activation of c-fos serum response element (SRE) and transformation in Rat-2 fibroblasts. Using a c-fos SRE-luciferase reporter gene, we showed that the transactivation of SRE by Ha-Ras(V12) is mainly via a Rac-linked cascade, although the Raf-mitogen-activated protein kinase cascade is required for full activation. In addition, Ha-Ras(V12)-induced DNA synthesis was significantly attenuated by microinjection of recombinant Rac(N17), a dominant negative mutant of Rac1. To identify the mediators downstream of Rac in the Ha-Ras(V12) signaling, we investigated the involvement of cytosolic phospholipase A(2). Oncogenic Ha-Ras(V12)-induced SRE activation was significantly inhibited by either pretreatment with mepacrine, a phospholipase A(2) inhibitor, or cotransfection with the antisense oligonucleotide of cytosolic phospholipase A(2). We also found cytosolic phospholipase A(2) to be situated downstream of Ha-Ras(V12) in a signal pathway leading to transformation. Together, these results are indicative of mediatory roles of Rac and cytosolic phospholipase A(2) in the signaling pathway by which Ha-Ras(V12) transactivates c-fos SRE and transformation. Our findings point to cytosolic phospholipase A(2) as a novel potential target for suppressing oncogenic Ha-Ras(V12) signaling in the cell.

Role of Protein Kinase Cζ in Ras-mediated Transcriptional Activation of Vascular Permeability Factor/Vascular Endothelial Growth Factor Expression

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is regulated by different factors including degree of cell differentiation, hypoxia, and certain oncogenes namely, ras and src. The up-regulation of VPF/VEGF expression by Ras has been found to be through both transcription and mRNA stability. The present study investigates a novel pathway whereby Ras promotes the transcription of VPF/VEGF by activating protein kinase Czeta (PKCzeta). The Ras-mediated overexpression of VPF/VEGF was also found to be inhibited by using the antisense or the dominant-negative mutant of PKCzeta. In co-transfection assays, by overexpressing oncogenic Ha-Ras (12 V) and PKCzeta, there was an additive effect up to 4-fold in activation of Sp1-mediated VPF/VEGF transcription. It has been shown through electrophoretic mobility shift assay that Ras promoted the PKCzeta-induced binding of Sp1 to the VPF/VEGF promoter. In the presence of PDK-1, a major activating kinase for PKC, the Ras-mediated activation of VPF/VEGF promoter through PKCzeta was further increased, suggesting that PKCzeta can serve as an effector for both Ras and PDK-1. In other experiments, with the use of a dominant-negative mutant of phosphatidylinositol 3-kinase, the activation of VPF/VEGF promoter through Ras, PDK-1, and PKCzeta was completely repressed, indicating phosphatidylinositol 3-kinase as an important component of this pathway. Taken together, these data elucidate the signaling mechanism of Ras-mediated VPF/VEGF transcriptional activation through PKCzeta and also provide insight into PKCzeta and Sp1-dependent transcriptional regulation of VPF/VEGF.

Comparison of early onco/suppressor gene expressions in peripheral leukocytes and potential target organs of rats exposed to the carcinogen 1-nitropyrene.

An in-vivo model has been developed to study early expressions of c-myc, Ha-ras oncogenes and p53 suppressor gene as biomarkers of carcinogenic exposure and/or tumorigenesis. In order to validate the in-vivo expression changes as biomarkers, rats were treated with the outdoor air pollutant carcinogen 1-nitropyrene. The gene expression levels were measured after 24 and 48 h in potential target tissues (lung, liver, lymph nodes, kidneys, spleen) and in peripheral blood leukocytes. Another main objective was to prove the applicability of leukocytes as a surrogate tissue, having a similar expression pattern of the selected genes upon carcinogenic exposure. The c-myc oncogene was not suitable as an early biomarker because of the lack or low level of its expression. However, in the case of the other oncogene Ha-ras and the suppressor gene p53, remarkable and early changes were detected in the expression signals. Similar expression patterns could only be detected in leukocytes and the spleen; therefore we continue this validation study by using other types and routes of exposure.

Theophylline and cisplatin synergize in down regulation of BCL-2 induction of apoptosis in human granulosa cells transformed by a mutated p53 (p53 val135) and Ha-ras oncogene.

Cisplatin is in common use in ovarian cancer therapy, although it is also implicated in cytotoxicity in normal tissue. We have examined the effect of cisplatin alone and in combination with theophylline, a phoshodiesterase inhibitor, on modulation of Bcl-2/Bax expression and induction of apoptosis in human granulosa cells transformed by stable transfection with mutant p53 plus Ha-ras. Theophylline elicited cell death only at relatively high concentrations with an EC50 of 200 microg/ml. Cisplatin exerted its lethal effect with an EC50 of 7 microM. In the presence of 15 or 50 microg/ml of theophylline (in the range used against asthma in humans), the EC50 for cisplatin was reduced to 2 microM or 1.2 microM, respectively. Using fluorescence-activated cell sorting analysis of DNA stained cells and the terminal deoxy-nucleotide tranferase-mediated dUTP nick end-labeling method, we found that even at concentrations of 0. 3 and 1 microM cisplatin, theophylline at 15 and 50 microg/ml increased the incidence of apoptosis in these cells by 3-5-fold, while theophylline alone induced extremely low apoptosis. Neither drug had any measurable effect on Bax protein expression. In contrast Bcl-2 protein expression levels were markedly reduced by theophylline and cisplatin in a dose-dependent manner. The combination of theophylline and cisplatin resulted in a further dramatic reduction in Bcl-2, under-scoring the pronounced synergy of these two drugs. These observations suggest that suppression of Bcl-2 expression may play an important role in mediating the synergistic effect of cisplatin and theophylline on induction of apoptosis in ovarian cancer cells.

Identification of a Novel Ligand-Receptor Pair Constitutively Activated by ras Oncogenes

The Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target, mob-5, encoding a 23-kDa cytokine-like secreted protein. Mob-5 expression could be induced by oncogenic Ha-ras and Ki-ras, but not by normal ras activation. Inhibitors of both Ha-Ras and mitogen-activated protein kinase kinase completely abolished the mob-5 expression in ras transformed cells, with concomitant loss of the transformation phenotype. Using an alkaline phosphatase-tagged Mob-5 as ligand, a putative Mob-5 receptor was identified on the cell surface of oncogenic ras transformed cells. Thus, the Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by ras oncogenes.

Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explanted DMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19(ARF), Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARF and functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null and ARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1(+/+) animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha- ras -transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G1 arrest that lasted ~16–24 h (IC50 ~0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3–7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor β (TGFβ) from the culture medium, supplementing the culture medium with anti-TGFβ or soluble TGFβII receptor, or pre-absorption of conditioned medium with anti-TGFβ all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by ~50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA ~50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for ~72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGFβ supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGFβII receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

S-Nitrosocysteine Increases Palmitate Turnover on Ha-Ras in NIH 3T3 Cells

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.

Expression of Oncogenic Epidermal Growth Factor Receptor Family Kinases Induces Paclitaxel Resistance and Alters β-Tubulin Isotype Expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.