NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.     

ras Transfection and expression does not induce progression from tumorigenicity to metastatic ability in mouse LTA cells

Studies testing the ability of a transfected ras oncogene to confer metastatic properties on non-metastatic cells have yielded conflicting results. Most of these studies have used recipient cells at early stages of progression (primary or immortalized, non-tumorigenic lines). In this study we tested the ability of the T24-H-ras oncogene to induce progression of tumorigenic, non-metastatic, murine LTA cells to a metastatic phenotype. Metastatic ability was assessed in complementary assays in two immune-deficient hosts, nude mice (after s.c. injection) and chick embryos (after i.v. injection), to determine if ras transfection affected metastatic properties in hosts lacking an intact immune system. Even with greatly elevated levels of ras p21 protein, pools of ras-transfected cells as well as individual clonal populations remained non-metastatic in both hosts. Serial in vivo passaging did not consistently enhance for either ras expression or metastatic ability. We conclude that expression of an activated ras oncogene in LTA cells does not induce progression from a tumorigenic to a metastatic phenotype. These results are in marked contrast to those obtained for ras expression in most other cell types. High levels of expression of an activated ras oncogene thus do not always promote progression from tumorigenicity to metastatic ability.     

Expression of oncoproteins in primary human non-small cell lung cancer and incidence of metastases

In the current study the relationship between the incidence of metastatic spread and expression (at the protein level) of various proto-oncogenes was investigated in 217 human non-small cell lung carcinomas. Tumors with an overexpression of proteins encoded by the oncogenes c-jun and c-myc showed a significantly increased formation of metastases (c-jun: P = 0.008; c-myc: P = 0.018). No significant correlations were found between the expression of the c-fos, c-erbB1, c-neu and c-ras products and metastatic spread.     

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8⁺ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4–12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K^d; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8⁺ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4⁺ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2K^d was demonstrated with the mutant ras4–12(V12) peptide, but not the normal ras4–12(G12) peptide, which specifically inhibited an H-2K^d-restricted, anti-nucleoprotein NP147–155 CTL response in a dose-dependent fashion. An anti-ras CD8⁺ T cell line was then established from immune splenocytes of BALB/c (H-2^d) mice injected with ras4–12(V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2K^d and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly → Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras 4–12(V12) peptide was immunogenic for the production of antigen-specific CD8⁺ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.     

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Chromosomal location and structure of amplicons in two human cell lines with coamplification of c-myc and Ki-ras oncogenes

Gene amplification is a major mechanism through which oncogenes and genes responsible for drug resistance are overexpressed in neoplastic cells, and several models for structure of amplified units (amplicons) are postulated. In order to identify consistent changes associated with oncogene amplification, we analyzed chromosomal location and physical distance of amplicons of two independent human cell lines that have coamplified c-myc and Ki-ras oncogenes. In one cell line, KHC287, amplified c-myc genes were localized in two chromosomes and Ki-ras in three chromosomes. One marker chromosome was almost entirely encompassed by both amplified genes. In the other cell line, Lu-65, both of the amplified genes shared the same locus, on chromosome 12q+. The two genes, however, are more than 1500 kb apart in both cell lines. The above findings indicate that two different amplified genes became associated on one chromosome in two independent cell lines. This suggests that a common mechanism is associated with chromosomal rearrangements affecting different amplified genes.     

Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4⁺ T helper type 1 (T_h1) subset. BALB/c mice (H-2^d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An αβ T cell receptor-positive, CD3⁺, CD4⁺, CD8^? T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-γ, tumor necrosis factor and granulocytemacrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Ia^d-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4⁺-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II^? P815 targets, inhibition of A20 lysis with anti-Ia^d monoclonal antibodies, and induction of lysis against L cell targets transfected with EαAβ^d. Independent isolation of a second CD4⁺ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4⁺ T_h1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.     

Someras-transformed cells have increased radiosensitivity and decreased repair of sublethal radiation damage

We examined the effect of⁶⁰Co irradiation on the clonogenic survival of rat NRK cells, NRK cells carrying a temperature-sensitive viralK-ras oncogene (tsK-NRK), mouse NIH 3T3 cells, and NIH 3T3 cells transformed with the human bladder cancer (T24)H-ras oncogene (PAP2). We tested the hypothesis thatras oncogene expression renders cells more resistant to radiation, but found in both systems thatras-transformed cells were more, not less, sensitive to radiation. We also found indications of altered repair of sublethal radiation damage. PAP2 cells were more sensitive to radiation than NIH 3T3 cells. Increased sensitivity was reflected in a decreased shoulder region of the survival curve with little effect on its slope (D ₀). TsK-NRK cells were also slightly more sensitive to radiation than NRK and exhibited decreased repair of sublethal damage at both the permissive and nonpermissive temperatures. Thus, we found that expression ofras oncogenes is not always associated with increased radiation resistance. In summary, our results suggest that (1)ras oncogene expression in some cells may be associated with increased, rather than decreased, radiation sensitivity, and (2)ras oncogene expression may alter the shoulder region of the does response curve, suggesting changes in the repair of sublethal radiation damage.     

Expression ofRap 1 suppresses genomic instability ofH-ras transformed mouse fibroblasts

Among the multiple genetic changes that occur during cancer progression are the activation of proto-oncogenes and the inactivation or loss of genes encoding tumor suppressors. The potential roles for these genes in the perturbation of genome stability continues to be of major interest. We have previously shown that conditional expression of H-ras in NIH3T3 cells increases genetic instability in these cells, rendering them more permissive to gene amplification and to the generation of chromosome aberrations which can be induced within a single cell cycle. In the present study we show that genetic instability induced by H-ras expression can be suppressed by co-expressions ofRap 1, aRas-related tumor suppressor gene. An NIH3T3 cell line transformed with activated human H-ras was transfected withRap 1. Expression of theRap 1 gene reverted the transformed cells to a flat morphology. The reverted cells reestablished contact inhibition of growth and lost the capacity to form colonies in soft agar. These cells were subsequently studied for the role ofRap 1 on the suppression of genomic instability induced by oncogenic H-ras. Cells transformed with H-ras manifest an increase in methotrexate resistance as measured by an increase inDhfr gene amplification. Cells which concommitantly expressRap 1 showed reduced levels of methotrexate resistance as well as reduction of gene amplification capacity. Furthermore fluorescent-in-situ hybridization (FISH) with a pancentromeric mouse probe showed that elevated levels of chromosome aberrations in cells expressing H-ras were also suppressed after co-expression ofRap 1.     

Ras levels and metalloproteinase activity in normal versus neoplastic rat mammary tissues

We have previously reported that activatedras oncogenes can simultaneously switch on the metastatic phenotype and increased capability to degrade type IV collagen [36]. Here the relationship between c-H-ras, metalloproteinase expression and metastatic behavior was studied inN-nitrosomethylurea (NMU)-induced rat mammary carcinomas, which are known to possess activated c-H-ras. When comparing normal rat breast tissue to mammary carcinomas there was no direct relationship betweenras DNA levels and neoplastic changes. Furthermore, there were no consistent differences between metastatic and non-metastatic carcinomas, or between primary tumors and metastases. The NMU-induced rat mammary carcinomas expressed two major gelatinolytic metal-loproteinases (gelatinases) of 65 and 92kD, but only the 65kD gelatinase was detected in normal breast tissue and a rat fibroma. Type IV collagenolytic activity per 5 g of protein was two to three times higher in the mammary carcinomas than in the normal breasts, whereas the primary tumors did not differ from the corresponding metastases. This study shows thatras amplification is not necessary for development of the malignant or metastatic phenotype in the NMU-induced rat mammary carcinoma model. We have also found that induction of p21ras protein synthesis in a v-H-ras transfected NIH/3T3 (433) cell line, containing a glucocorticoid promoter, does not lead to an increase in metastatic capacity.     

Cell shrinkage stimulates bradykinin-induced cell membrane potential oscillations in NIH 3T3 fibroblasts expressing the ras-oncogene

In NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) bradykinin leads to sustained oscillations of cell membrane potential due to oscillations of intracellular Ca²⁺ with subsequent activation of Ca²⁺-sensitive K⁺ channels. In cells not expressing the oncogene (-ras), bradykinin leads only to a single transient hyperpolarization of the cell membrane. The present study has been performed to elucidate the possible interaction of cell volume, intracellular pH and bradykinin-induced oscillations of the cell membrane potential. Bradykinin leads to cell shrinkage and intracellular alkalinization of both +ras cells and –ras cells. Inhibition of Na⁺/H⁺ exchanger by HOE 694 abolishes the bradykinin-induced alkalinization but does not significantly interfere with the bradykinin-induced oscillations of cell membrane potential. In contrast, prevention of bradykinin-induced cell shrinkage by simultaneous reduction of extracellular osmolarity blunts the oscillations. Thus, cell shrinkage stimulates bradykinin-induced oscillations of cell membrane potential. On the other hand, cell shrinkage alone does not elicit oscillations unless, in addition, Ca²⁺ entry is stimulated by ionomycin.     

Tumorigenicity,invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA

Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants,in vitro derivatives, andin vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examinedin vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was testedin vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasivein vitro and tumorigenicin vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passagein vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.     

No correlation of c-myc overexpression and p53 mutations in liposarcomas

 Although it is well known that oncogenesis is a multistep process involving the activation of normal cellular genes to become oncogenes and/or the inactivation of tumor suppressor genes, this process has seldom been investigated in soft tissue tumours. We screened a group of 36 liposarcomas for genetic abnormalitis in the p53 tumour suppressor gene and c-myc oncogene. Altered c-myc gene expression was examined by differential RT-PCR assay. p53 Gene mutations in exons 4–8 were analysed by using PCR-SSCP analysis and direct sequencing. Elevated c-myc expression was found in 6 of 31 liposarcomas (19.4%). p53 Gene mutations were observed in 5 of 36 liposarcomas (13.9%). Both genetic alterations were associated with the histological subtype of liposarcomas. Whereas c-myc gene expression was a characteristic of myxoid/round cell liposarcomas, p53 gene mutations were found more frequently in pleomorphic variants. Liposarcomas of the well-differentiated subtype showed neither p53 gene mutations nor altered c-myc gene expression. Our results indicate that the c-myc oncogene and the p53 tumor suppressor gene do not seem to cooperate in the oncogenesis of liposarcomas. Received: 22 April 1998 / Accepted: 11 May 1998     

Acquisition and enhanced expression of the metastatic phenotype following transfections of genomic mouse tumor DNA containing human SCLC gene sequences

Previous primary and secondary co-transfections of genomic DNA from a metastatic human small cell lung cancer cell line into NIH/3T3 cells resulted in a murine fibrosarcoma cell line (Tx93B) that produced frequent spontaneous lung metastases in subcutaneously injected tumor-bearing nude mice. In order to transfer the acquired metastatic behavior to additional cell lines that could then be tested in syngeneic immunocompetent animals, DNA from Tx93B cells was transfected without additional neo gene into Balb/c embryo fibroblasts, which led to the isolation of a tertiary transfectant cell line (D3) of low spontaneous metastatic potential in normal Balb/c mice. Subsequent cell lines established serially from lung metastases in mice injected with D3, and metastatic descendants of D3 (all selected for the original neo marker in G-418), resulted in three generations of metastatically variant cell lines capable of causing pulmonary metastases in 11.1%, 54.6%, and 89.5%, respectively, of subcutaneously injected animals, and in 100% of normal mice injected intraperitoneally. There was no apparent ras-family oncogene participation in the metastatic behavior of either of the two DNA donor cell lines or in the metastatically variant tertiary transfectants. Gelatin zymography indicated that the secretion of gelatinolytic enzymes in vitro by the variant cell lines was inversely proportional to their metastatic capability. Human Alu repeat gene sequences detected in the metastatic variants suggested that co-transfected metastasis-associated genes present in the original human DNA donor cell may have contributed to acquisition of the metastatic phenotype by the tertiary transfectant cell lines. The increase in metastatic potential observed in successive generations of the D3-derived tumor cell lines, further suggested that selection for cells having increased metastatic capability had occurred during passage in vivo accounting for the phenotypic change. Because of their common origin and progressively metastatic nature these cell lines may prove useful in the identification of metastasis-associated genes accessible through the use of differential expression cloning strategies.     

A PSP94-derived Peptide PCK3145 inhibits MMP-9 Secretion and Triggers CD44 Cell Surface Shedding: Implication in Tumor Metastasis

Purpose: PCK3145 is a synthetic peptide corresponding to amino acids 31–45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. Experimental design: HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). Results: We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. Conclusions: Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor. *These authors contributed equally to this work.     

Multiple genetic alterations in malignant metastatic insulinomas

Proto-oncogenes, growth factors/receptors, and tumour suppressor genes were analysed in malignant metastatic insulinomas. Normal pancreas showed only a moderate immunoreaction for c-myc proto-oncogene and a strong reaction for insulin. Benign insulinomas were slightly or moderately positive for transforming growth factor a (TGFα), weakly positive for epidermal growth factor receptor (EGF-R), and strongly positive for c-myc and insulin. In malignant insulinomas, besides a strong immunoreaction for c-myc and TGFα, activation of c-K-ras and overexpression of p53 protein were found. Insulin reaction was moderate or strong. Three out of six malignant insulinomas displayed a c-K-ras point mutation at codon 12. All mutations were guanine to cytosine transversion, resulting in amino acid substitution, glycine to arginine. Mutations were present in metastatic insulinomas only. Patients with mutated c-K-ras oncogene had overexpression of p53 protein as well as c-myc and TGFα overexpression. Our results support the view that malignant progression is a consequence of more than one genetic lesion and suggest that activation of myc, TGFα, and ras genesα plays a role in a multistep process of tumour progression, perhaps serving as an initiating event.     

Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells

The standard, 85–95-kDa form of the hyaluronic acid (HA) receptor CD44 and a number of CD44 mRNA splice variants play important roles in immune responses and tumor metastasis. Variants carrying exon 6 (v6), or 9 (v9) products are transiently expressed on activated human T cells. Here, modulation experiments with specific monoclonal antibodies (mAb) indicate that v6 and v9 are expressed independently on distinct sets of CD44 molecules, and that their combined expression is necessary for HA adhesion. Moreover, the finding that mAb-mediated cross-linking of v6 and v9 promoted cytosolic free Ca²⁺ mobilization and co-stimulated CD3-triggered T cell proliferation indicates that v6 and v9 possess signaling and effector function activation ability. Finally, HA-mediated signaling appears to be required for variant-dependent adhesion to HA. The observation that soluble HA promoted cytosolic free Ca²⁺ mobilization indicates that HA-induced Ca²⁺ mobilization can occur during T cell-HA interaction. Since Ca²⁺ mobilization was inhibited by pretreatment of cells with an anti-CD44 mAb directed against the HA-binding domain of CD44, CD44 receptors appear to be involved in HA-mediated signal transduction. The requirement of cytosolic free Ca²⁺ for adhesion is shown by the fact that ionomycin (a Ca²⁺ ionophore) stimulated, and EGTA (a Ca²⁺ chelator), inhibited HA adhesion. In addition, cytoskeletal functional activation is required for cell adhesion to HA, since drugs that block actin polymerization, such as cytochalasin B, or actomyosin contraction, such as the calmodulin antagonist W-7, inhibited cell adhesion to HA. As this adhesion is also ADP ribosylation-sensitive, it may involve a GTP-dependent function of CD44v, i.e. ankyrin binding. Our data indicate that there is a functional hierarchy among the CD44 molecules expressed on human peripheral blood T cells and that the splice variants, as compared to the standard form, exhibit a greater HA binding ability which involves CD44-mediated signaling and effector function activation.     

Suppression of serum-induced c-jun expression by activated Ki-ras in human colon cancer cells

Summary Through gene-targeting, we have established human colon cancer cell lines, HK2-6 and HKe-3, with and without activated Ki-ras, respectively, derived from a human colon cancer cell line HCT116, and we have reported that activated Ki-ras is involved in the deregulation of c-myc expression. To further examine the relation between Ki-ras-mediated signals and other immediate early genes, c-jun was analyzed on these cells stimulated by serum. Rapid and strong induction of c-jun was observed in HKe-3, but not in HCT116 or HK2-6. To elucidate the regulatory mechanisms of c-jun expression by Ki-ras, protein kinase C (PKC) and c-Raf were examined at serum-starved and serum-stimulated conditions. Phosphorylations of c-Raf were same among these cells, however, the cytosolic PKC activity in HKe-3 was two times higher than that in HCT116 on serum-starved and serum-stimulated conditions. These results suggested that serum responsiveness of c-jun may be suppressed by activated Ki-ras through PKC rather than c-Raf pathway in colon cancer cells.     

A search for activating N-ras mutations in malignant histiocytosis of the bernese mountain dog

Activated ras alleles have been detected in a wide range of human neoplasms, and c-N-ras mutations predominate in haematopoietic neoplasms. Ras oncogenes have been implicated in several animal and human models of multistep carcinogenesis. Malignant histiocytosis is a rare neoplasm of dogs that appears unique to the Bernese Mountain Dog. In the present study, we have examined the c-N-ras gene in two normal and 16 neoplastic Bernese Mountain Dog tissues by a polymerase chain reaction sequencing strategy. No activation of c-N-ras alleles was detected at codons 12, 13 or 61- those sites for ras proto-oncogene activations in human neoplasms. Indeed, in those regions of the first and second exons of canine c-N-ras examined, the nucleotide sequences derived from malignant histiocytosis specimens were identical to those derived from normal tissues, and the Bernese Mountain Dog sequence was identical to that which we have previously described in the Beagle dog. c-N-ras mutation does not appear to be associated with the aetiopathogenesis of malignant histiocytosis in the Bernese Mountain Dog.