The highly lipophilic DNA topoisomerase I inhibitor DB-67 displays elevated lactone levels in human blood and potent anticancer activity.

The novel silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67) is 25- to 50-times more lipophilic than camptothecin and readily incorporates into lipid bilayers. Using the method of fluorescence anisotropy titration, we determined that DB-67 bound to small unilamellar vesicles composed of dilaurylphosphatidylcholine (DLPC) with an association constant (K value) of 5000 M(-1). This association constant is significantly higher than the K(DLPC) value observed for camptothecin (K(DLPC) value of 110 M(-1)). Using HPLC methods, we demonstrated that the presence of liposomal membranes readily stabilize the lactone form of DB-67. At drug and lipid concentrations of 10 microM and 0.3 mM, respectively, the lactone form of DB-67 persisted in liposome suspension after 3 h of incubation at 37 degrees C. Thus an advantage of a liposomal formulation of DB-67 is that the presence of lipid bilayers assists with stabilizing the key pharmacophore of the agent. The highly lipophilic character of DB-67, in combination with its 10-hydroxy moiety (which functions to enhance lactone stability in the presence of human serum albumin), results in DB-67 having superior stability in human blood with a percent lactone at equilibrium value of 30 [Cancer Res. 59 (1999) 4898; J. Med. Chem. 43 (2000) 3970]. Potent cytotoxicities against a broad range of cancer cells were observed for DB-67, indicating that DB-67 is of comparable potency to camptothecin. The impressive human blood stability and cytotoxicity profiles for DB-67 indicate it is an excellent candidate for comprehensive in vivo pharmacological and efficacy studies. Based on these promising attributes, DB-67 is currently being developed under the NCI RAID program. Due to its potent anti-topoisomerase I activity and its intrinsic blood stability, DB-67 appears as an attractive novel camptothecin for clinical development.

The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis.

We have shown elsewhere that equine-2 influenza virus (EIV; subtype H3N8) induced pronounced cell death in infected cells through apoptosis as demonstrated by DNA fragmentation assay and a combined TUNEL and immunostaining scheme. In this study, we investigated the mechanism of EIV-mediated cytotoxicity on a permissive mammalian epithelial cell line, Madin-Darby canine kidney (MDCK) cells. EIV infection increased the cellular levels of oxidative stress and c-Jun/AP-1 protein (which is known to be affected by oxidative stress), as well as its DNA binding activity. Increased production of TGF-beta1, an inducer of c-Jun N-terminal kinase or stress-activated protein kinase (JNK/SAPK) activation, was also detected in EIV-infected MDCK cells. It has been reported that TGF-beta may initiate a signaling cascade leading to JNK/SAPK activation. Addition of c-Jun antisense oligodeoxynucleotide, antioxidant N-acetyl-cysteine (NAC), JNK/SAPK inhibitor carvedilol, or TGF-beta-neutralizing antibody effectively blocked c-Jun/AP-1 upregulation and TGF-beta1 production mediated by EIV infection. These treatments also attenuated EIV-induced cytopathogenic effects (CPE) and apoptosis. Our results suggest that a stress-activated pathway is involved in apoptosis mediated by EIV infection. It is likely that EIV infection turns on the JNK/SAPK cascade, which modulates the activity of apoptosis-promoting regulatory factor c-Jun/AP-1 and epithelial growth inhibitory cytokine TGF-beta.

Histone deacetylase inhibitors promote apoptosis and differential cell cycle arrest in anaplastic thyroid cancer cells.

Little information exists concerning the response of anaplastic thyroid carcinoma (ATC) cells to histone deacetylase inhibitors (HDAIs). In this study, the cellular response to the histone deacetylase inhibitors, sodium butyrate and trichostatin A, was analyzed in cell lines derived from primary anaplastic thyroid carcinomas. HDAIs repress the growth (proliferation) of ATC cell lines, independent of p53 status, through the induction of apoptosis and differential cell cycle arrest (arrested in G1 and G2/M). Apoptosis increases in response to drug treatment and is associated with the appearance of the cleaved form of the caspase substrate, poly-(ADP-ribose) polymerase (PARP). Cell cycle arrest is associated with the reduced expression of cyclins A and B, the increased expression of the cyclin-dependent kinase inhibitors, p21(Cip1/WAF1) and p27Kip1, the reduced phosphorylation of the retinoblastoma protein (pRb), and a reduction in cdk2 and cdk1-associated kinase activities. In ATC cells overexpressing cyclin E, drug treatment failed to replicate these events. These results suggest that growth inhibition of ATC cells by HDAIs is due to the promotion of apoptosis through the activation of the caspase cascade and the induction of cell cycle arrest via a reduction in cdk2- and cdk1-associated kinase activities.

Butyrate alters the expression and activity of cell cycle components in anaplastic thyroid carcinoma cells.

Anaplastic thyroid carcinoma (ATC) is the most malignant and aggressive form of thyroid cancer. Most patients die within months of diagnosis, primarily due to the absence of effective chemotherapeutic strategies. Identifying alternative therapies is necessary to increase long-term survival. Butyrate elicits a number of responses from cancer cells both in vitro and in vivo including growth repression, cell cycle arrest, differentiation, and apoptosis. Even though many types of cancer cells have been studied, little is known of the response of ATC cells to this drug. In this study, we report that butyrate induces differential cell cycle arrest (arrest in G1 and G2/M phases) in an ATC cell line that correlates with changes in the expression, phosphorylation, and activity of key components of the cell cycle machinery. Exposure to butyrate increases the expression of the cyclin-dependent kinase inhibitors, p21/Cip1 and p27/Kip1, decreases the expression of cyclin A and cyclin B, inhibits the phosphorylation of the retinoblastoma protein (pRb), and decreases the activity of cdk1 and cdk2-associated kinases. These results suggest that butyrate may be useful in the clinical treatment of ATC.

The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and potent anticancer activity.

We describe the rational design and synthesis of B- and A, B-ring-modified camptothecins. The key alpha-hydroxy-delta-lactone pharmacophore in 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, 14) displays superior stability in human blood when compared with clinically relevant camptothecin analogues. In human blood 14 displayed a t(1/2) of 130 min and a percent lactone at equilibrium value of 30%. The tert-butyldimethylsilyl group renders the new agent 25-times more lipophilic than camptothecin, and 14 is readily incorporated, as its active lactone form, into cellular and liposomal bilayers. In addition, the dual 7-alkylsilyl and 10-hydroxy substitution in 14 enhances drug stability in the presence of human serum albumin. Thus, the net lipophilicity and the altered human serum albumin interactions together function to promote the enhanced blood stability. In vitro cytotoxicity assays using multiple different cell lines derived from eight distinct tumor types indicate that 14 is of comparable potency to camptothecin and 10-hydroxycamptothecin, as well as the FDA-approved camptothecin analogues topotecan and CPT-11. In addition, cell-free cleavage assays reveal that 14 is highly active and forms more stable top1 cleavage complexes than camptothecin or SN-38. The impressive blood stability and cytotoxicity profiles for 14 strongly suggest that it is an excellent candidate for additional in vivo pharmacological and efficacy studies.

Translational control of malignancy: the mRNA cap-binding protein, eIF-4E, as a central regulator of tumor formation, growth, invasion and metastasis.

Recent studies have implicated the mRNA cap-binding protein, eIF-4E, as a key regulator of malignant progression. Indeed, the major intracellular signaling pathways involved in tumor growth and malignancy, the MAP kinase and PI3 kinase pathways, induce eIF-4E activity. Furthermore, immunohistochemical analyses have revealed that eIF-4E is overexpressed and related to disease progression in human cancers of the colon, head and neck, and breast. In experimental tumors, manipulation of eIF-4E function profoundly affects not only tumorigenesis but also tumor invasion and metastasis. While increasing global protein synthesis rates, the increased activity of eIF-4E that typifies both human and experimental tumors disproportionately enhances the translation of a specific array of potent growth regulatory and malignancy-related proteins, including c-myc, cyclin D1, ornithine decarboxylase, vascular endothelial growth factor, basic fibroblast growth factor and others. Herein, we review the data supporting the notion that, by coordinately upregulating the translation of numerous malignancy-related proteins, eIF-4E plays a pivotal role in regulating not only tumor growth, but also invasion and metastasis.

A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell.

Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF alpha-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).

Two new human neuroblastoma cell lines exhibiting tumorigenesis and metastasis in the nude mouse model.

BACKGROUND: Human neuroblastoma cell lines are notoriously difficult to establish in culture and use in murine hosts. MATERIALS AND METHODS: Two new human neuroblastoma cell lines, NK and ND, were established and studied for growth patterns in nude mice, growth in soft agar, cell cycle analysis, apoptosis (Hoechst- merocyanine 540 test), metalloproteinase expression (zymograms), and morphological differentiation by dibutyryl cyclic AMP (dCAMP). RESULTS: Both cell lines formed tumors in 6/9 nude mice within 5-31 days after subcutaneous inoculation, and metastases after intravenous tail vein injection. Both grew in soft agar. DCAMP induced morphologic differentiation in both, and inhibited cell culture growth without apoptosis. Zymograms of supernatants from cultures revealed 72-kDa metalloproteinase and higher molecular bands that did not change with dCAMP treatment. Cultures derived from murine metastatic foci exhibited 72, 82 and 85-kDa proteins, with strong 92-kDa bands after dCAMP treatment. CONCLUSION: New human neuroblastoma cell lines were established that are easily used in nude mice, and express metalloproteinases.

Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurthle's cell, and poorly differentiated human thyroid carcinoma.

Expression of the invasion/metastasis suppressor, E-cadherin, is diminished or lost in thyroid carcinomas. Yet, mutational inactivation of E-cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11% of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently compromised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

Translation of ODC mRNA and polyamine transport are suppressed in ras-transformed CREF cells by depleting translation initiation factor 4E.

Rapid tumor growth and metastasis require increased polyamine metabolism, which is coordinately regulated by ornithine decarboxylase (ODC) and the polyamine transporter. Both activities are stimulated by ras signalling and are dependent upon protein biosynthesis. T24ras oncogene expression in rat embryo fibroblasts (CREFT24) induces cellular transformation and malignancy, in part, by stimulating the rate-limiting translation initiation factor, eIF-4E. CREFT24 expressing antisense RNA to eIF-4E (AS4E) have markedly decreased tumor growth rates and metastatic capacity, without altered monolayer growth rates. Herein, we demonstrate that in AS4E, ODC is translationally suppressed resulting in decreased ODC activity. Additionally, exogenous polyamine uptake is suppressed in AS4E cells indicating that AS4E can neither generate nor import the polyamines necessary to support rapid tumor growth. These data provide evidence that eIF-4E is the link between ras-induced malignancy and increased polyamine metabolism and support the hypothesis that eIF-4E plays a pivotal role in mediating ras-induced malignancy.

Elevated expression of eIF4E and FGF-2 isoforms during vascularization of breast carcinomas.

The translation initiation factor eIF4E is a novel protooncogene found over expressed in most breast carcinomas (Kerekatte et al., 1995), but the pathology where this elevation is initially manifested and its possible role in cancer progression are unknown. We report that eIF4E is markedly increased in vascularized malignant ductules of invasive carcinomas, whereas necrotic and avascular ductal carcinomas in situ display significantly lower levels. eIF4E facilitates the synthesis of FGF-2, a powerful tumor angiogenic factor. Conversely, reducing eIF4E with antisense RNA in MDA-435 cells suppresses their tumorigenic and angiogenic properties, consistent with loss of FGF-2 synthesis. These findings suggest a causal role for eIF4E in tumor vascularization.

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell line in vitro and in vivo.

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experiments in vivo and in vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87 in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluation in vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DRO90-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membrane in vitro.

Phospholipases A2 in ras-transfected fibroblasts.

Phospholipase A2 (PLA2) generated lipid biomediators can facilitate neoplastic progression. Specific PLA2 alterations associated with ras oncogene expression were determined by comparison of PLA2 activities in nontumorigenic rat embryo fibroblasts (CREF cells) and their tumorigenic ras oncogene-transfected counterparts (CREF-T24 cells). The high molecular mass cytosolic PLA2 activity is 2-3 fold higher in CREF-T24 cells as compared to CREF cells. Western blotting analyses indicate increases in the level of this enzyme and the proportion which migrates with phosphorylated enzyme in CREF-T24 cells. A PLA2 activity, with the characteristics of a group II PLA2, is readily detectible in particulate fractions from CREF-T24 cells following ammonium sulfate extraction/cation ion exchange chromatography. In contrast, this activity is minimal in similarly prepared CREF cell samples. While the CREF-T24 cells have increases in two PLA2 activities associated with the release of arachidonic acid, the CREF-T24 and CREF cells are similar with respect to Ca2+ independent, particulate fraction-associated PLA2 activities.

Reduction of translation initiation factor 4E decreases the malignancy of ras-transformed cloned rat embryo fibroblasts.

Expression of the T24ras oncogene induces malignancy (tumor growth, invasion and metastasis) in cloned rat embryo fibroblasts (CREF T24). In CREF T24, the rate of phosphorylation of eukaryotic translation initiation factor 4E (eIF-4E) is increased, resulting in increased protein synthesis rates. We have recently shown that reducing the protein levels of eIF-4E in CREF T24 (AS4E line) markedly decreases soft-agar colonization, increases tumor latency periods and increases tumor doubling times without significantly altering monolayer growth. In this study, cells with reduced eIF-4E had delayed and reduced invasiveness and decreased experimental metastasis. Furthermore, reduced eIF-4E levels correlated with decreased expression of the metastasis-associated 92-kDa collagenase type-IV and exon-6 variants of the CD44 adhesion molecule [CD44(6v)]. Reduced eIF-4E levels correlated inversely with increased levels of the putative metastasis-suppressor protein nm23. Cell lines established from AS4E tumors and lung metastases exhibited increased levels of eIF-4E protein and protein synthesis rates compared to the AS4E line. Tumor-derived AS4E had the shortened tumor latency periods of CREF T24 but displayed the slow tumor-growth rates of AS4E. Tumor-derived AS4E exhibited the metastatic capacity of CREF T24 controls. Furthermore, tumor- and lung-nodule-derived AS4E expressed levels of CD44 (6v) and the 92-kDa collagenase type IV comparable to CREF T24 and displayed reduced levels of nm23 relative to AS4E. These results demonstrate that eIF-4E is an important effector molecule involved in oncogenic p21ras-induced malignant transformation.

Inhibition of collagenolytic activity relates to quantitative reduction of invasion in vitro in a c-Ha-ras transfected glial cell line.

The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissue in vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick hearts in vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the model in vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD. To verify whether gelatinase affected invasion in vitro of these glial cells we determined the efficacy of a substrate specific collagenase inhibitor on invasion in vitro. GM6001 is a synthetic polypeptide that specifically occupies the substrate binding sites of metalloprotease. Since this drug did not show cytotoxicity, its specificity for metalloprotease is a valuable tool to evaluate the physiological function of these enzymes on invasion. We found that treatment of CxT24neo3 with GM6001 reduced the fraction of invading CxT24neo3 cells through reconstituted basement membrane. These data suggest that metalloproteases can stimulate brain tumor invasion.

Decreasing the level of translation initiation factor 4E with antisense RNA causes reversal of ras-mediated transformation and tumorigenesis of cloned rat embryo fibroblasts.

Transformation of cloned rat embryo fibroblasts (CREF) with the T24-ras oncogene results in loss of contact inhibition, growth in soft agar and tumor formation in nude mice. Previously we showed that in such cells (CREF T24), the phosphorylation rate of protein synthesis initiation factor 4E (eIF-4E) is increased, correlating with an increase in the general rate of protein synthesis. In the present study, we have expressed antisense RNA complementary to eIF-4E mRNA in CREF T24 cells using a stably integrated vector. Cells expressing antisense RNA (CREF T24/AS) contained 30-50% of the normal level of eIF-4E and exhibited many of the properties of untransformed cells. CREF T24 had a spindle-shaped, refractile appearance, whereas CREF T24/AS grew in ordered, parallel patterns and exhibited contact inhibition similar to untransformed CREF. The rates of growth and protein synthesis in CREF T24/AS were decreased compared to CREF T24 but were not as low as in CREF. The efficiency of growth in soft agar was 11-fold lower for CREF T24/AS compared with CREF T24. The latency period for tumor formation in nude mice was increased from 8 days for CREF T24 to 17-27 days for CREF T24/AS and various clonal lines derived from them. Cell lines established from these CREF T24/AS-derived tumors were shown to have partially regained the eIF-4E levels characteristic of CREF T24. These results demonstrate that many of the phenotypic alterations associated with ras-induced malignant transformation can be reversed by a moderate reduction of the translational initiation capacity and therefore may be mediated through a translational mechanism.

Defining the critical gene expression changes associated with expression and suppression of the tumorigenic and metastatic phenotype in Ha-ras-transformed cloned rat embryo fibroblast cells.

Carcinogenesis requires a complex series of genetic changes often involving multiple oncogenes and the inactivation of multiple tumor-suppressor genes. We presently examined the effect of the Krev-1 tumor-suppressor gene on the tumorigenic and metastatic potential of Ha-ras-transformed cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed and anchorage independent; produce reduced levels of nm23-H1 (a putative metastasis-suppressor gene product) and TIMP-1 (tissue inhibitor of metalloproteinase 1) transcripts and mRNA compared with CREF cells; produce increased levels of cripto, 94-kDa gelatinase/type IV collagenase (94-kDa GEL), osteopontin (OPN) and transin/stromelysin transcripts and mRNA compared with CREF cells; and are tumorigenic and metastatic in both nude mice and syngeneic rats. Ha-ras-transformed CREF cells coexpressing the Krev-1 gene display a reversion in cellular phenotype and gene expression to that of untransformed CREF cells. However, Ha-ras/Krev-1-coexpressing CREF cells retain, albeit with extended latency periods, both tumorigenic and metastatic potential that is not related directly to the final level of Ha-ras or Krev-1 mRNA or the Ha-ras p21 transforming protein. Development of metastatic potential is, however, directly correlated with a reduction in nm23-H1 and TIMP-1 transcription and mRNA levels and an enhanced expression of cripto, 94-kDa GEL, osteopontin and transin. In contrast, expression of additional tumor-suppressor genes, such as the RB gene and p53, or genes associated with tumorigenesis in other model systems, such as major excreted glycoprotein (MEP), 72-kDa gelatinase/type IV collagenase (72-kDa GEL), fibronectin (FIB), tenascin and intracellular adhesion molecule 1 (ICAM-1) is not altered in a consistent manner during in vitro transformation suppression or escape from tumorigenic and metastatic suppression. These results indicate that Krev-1 suppression of the Ha-ras-transformed/oncogenic phenotype is associated with a distinct program of gene expression changes manifested by altered rates of transcription and steady-state mRNA levels of specific oncogenic-suppressing and oncogenic-inducing genes. These data support a model of Ha-ras-induced metastasis in CREF cells that involves a direct modulation in the expression/suppression of specific combinations of oncogenic-suppressor genes and metastasis-promoting genes that are regulated coordinately in the process of tumor progression.