Degradation of endothelial cell matrix collagen is correlated with induction of stromelysin by an activated ras oncogene

A conditional expression system was established whereby the human K-ras, v-src, and v-mos genes were cloned into a conditional expression vector downstream of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Rat-1 fibroblasts were transfected with these constructs and selected in medium containing G418. Cloned transfectants were isolated and characterized for absolute dependence on dexamethasone for expression of oncogene products and anchorage-independent growth in soft agar. Expression of activated p21^K-ras(val12) enabled the fibroblasts to degrade extracellular matrix collagen secreted by murine microvessel endothelial cells. Concurrent with p21^K-ras(val12) induction a proteinase with the characteristic size and substrate specificity of transin, the murine homologue of the human matrix metalloproteinase stromelysin, was expressed and secreted. Induction of v-mos and v-src oncogenes resulted in little or no detectable transin expression respectively coinciding with a relative or absolute failure to increase degradation of extracellular matrix collagen. This study suggests that in this system the expression of the ras oncogene can contribute to the in vitro invasive behavior of tumor cells by upregulating the production of a metalloproteinase capable of degrading collagen synthesized by vascular endothelial 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.     

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.     

Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence

Oncogene-dependent regulation and tumor relatedness of CD44 expression were investigated in Balb/c 3T3 cells and their derivatives transformed with different ras oncogenes (metastatic tumor model) or the human c-sis oncogene (non-metastatic model). Ras transformants using either the Harvey or Kirsten oncogenes expressed high levels of cell surface CD44 protein that bound fluoresceinated hyaluronan (HA). Much lower levels of CD44 were expressed in parental 3T3 cells, ras ⁻ revertants generated from Kirsten-transformed cells, or c-sis transformants, confirming the significance of the ras oncogene in this upregulation. To determine whether endogenous HA regulates these parameters, hyaluronidase treatment of ras transformants exposed more cell surface CD44 to anti-CD44 antibody and increased fluoresceinated HA binding; this did not occur with 3T3 or c-sis transformants. CD44 expression and its HA-binding function were conserved in a panel of in vivo primary and lung metastatic tumor cell lines derived from ras transformants. Ras transformants also retained the ability to downregulate CD44 protein levels in confluent cultures which occurred through a translational or post-translational mechanism (as CD44 mRNA levels were not reduced). These results taken together demonstrate that ras-dependent regulation of CD44 may correlate with tumor progression and metastasis in vivo, possibly (although not exclusively) supporting CD44's importance in metastatic progression.     

Enhanced expression of EGF receptor and low frequency of ras mutations in X-ray-induced rat thyroid tumours

Radiation is recognized as a carcinogenic factor for the thyroid gland. In this experimental study, oncogene expression was investigated in radiation-induced rat thyroid tumours. Forty 3-month-old Wistar rats received X-ray-irradiation to the neck region; 40 animals were untreated controls. After 14 months, thyroid tumours had developed in 25 of the 29 irradiated animals still alive; 76% of these tumours were considered malignant. No tumours developed in controls. Mutations of codons 12–13 and 59–63 of H-, K- and N-ras were analysed by PCR-SSCP (single-strand conformation polymorphism analysis) and sequencing of DNA from thyroid tissue. SSCP indicated a ras mutation frequency of 8%, but only one K-ras codon 12 (Gly-Cys) mutation was confirmed by sequencing. Protooncogene expression was analysed by mRNA slot blot hybridization analysis and immunohistochemistry. K-ras mRNA expression and EGF receptor mRNA and protein expression were significantly increased in the irradiated animals compared with controls, and in tumours versus nontumour tissue. This study of radiation-induced rat thyroid tumours demonstrates that ras expression may be subject to changes apart from activating mutations. Increased expression of EGF receptor in the tumours parallels the situation in human thyroid cancer. Received: 18 May 1999 / Accepted: 31 May 1999     

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.     

Transformed NIH 3T3 cells expressing human melanoma N-ras oncogene metastasize to lymph node in nude mice

The effect of the N-ras oncogene on the propensity of transformed cells to disseminate from the tumor and to metastasize, using NIH 3T3 cells transformed either with human melanoma DNA containing the N-ras oncogene or with the cloned N-ras from human neuroblastoma, was investigated. The results show that NIH 3T3 expressing these genes readily formed tumors after subcutaneous injection in nude mice. Spontaneous lymph node metastasis was observed after a first cycle of transfection in one animal inoculated with cells containing human melanoma N-ras oncogene, and in 95 per cent of the animals after the second and third rounds of transfection, indicating that the metastatic capacity was transferred. In all cases human N-ras oncogene was found in both the metastases and the associated tumors. No control NIH 3T3 cells formed tumors or metastases in nude mice, and NIH 3T3 cells transfected with cloned N-ras activated oncogene formed tumors in 100 per cent of injected mice, but no spontaneous metastases. Thus human activated N-ras gene may not be sufficient to confer metastatic behavior in nude mice and the metastatic ability of human melanoma DNA transfected cells may be due to, among other possibilities, expression of other gene sequences from melanoma DNA co-transfected with the N-ras oncogene, or to specific activated murine sequences switched on during the initial process of transfection.     

Tumor cell surface β1–6 branched oligosaccharides and lung metastasis

NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the1–6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in1–6 branched Asn-linked oligosaccharides.     

Cyclic AMP decreases chemotaxis,invasiveness and lung colonization of H-ras transformed mouse fibroblasts

We transfected mouse 10T1/2 fibroblasts with the H-ras oncogene and isolated lines expressing H-ras. One of the lines exhibited a highly malignant phenotype with the ability to produce large tumors and to colonize the lung after tail vein injection. In addition, the cells of this line showed increased collagenase IV production, directed migration and invasiveness, properties associated with the ability of tumor cells to metastasize. Since cyclic adenosine 3,5-monophosphate (cAMP) is known to down-regulate ras expression, we exposed the malignant cells (Cl-1) to either N⁶, 2,0-dibutyryl cAMP (DB-cAMP) or 8-bromo cAMP (8-Br-cAMP), either with or without a phosphodiesterase inhibitor. We found that these treatments reduced the expression of ras, chemotaxis, invasiveness, and lung colonization of the ras-transformed cells. We therefore postulate that the malignancy of some cells may be regulated by alterations in the intracellular cAMP levels by suppressing ras expression and/or by reducing other activities required for the dissemination of tumor cells.     

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.     

Lewis lung carcinoma (3LL) cells treated in vitro with ultraviolet radiation show reduced metastatic ability due to an augmented immunogenicity

The metastatic ability of 3LL tumor following in vitro irradiation with ultraviolet (u.v.) light was studied. Tumor cells were exposed to two courses of u.v.-irradiation (3LL × 2u.v. cells) and after two weeks of culture they were inoculated intravenously (i.v.) into syngeneic mice. These cells produced significantly fewer pulmonary metastases than the untreated population. In addition, intrafootpad (i.f.p.) injections of 3LL × 2u.v. cells into immunocompetent animals induced tumors only in 40 per cent of recipients. Interestingly, in normal mice with progressively growing 3LL × 2u.v. tumors, the formation of spontaneous pulmonary metastases was prevented, whereas metastatic foci were observed in 70 per cent of the nude recipients. The metastatic properties of u.v.-treated tumor cells were further analysed by using individual clones with varying immunogenicity. We found that variants with augmented immunogenicity also showed a parallel decrease in metastatic potential. Studies on H-2 antigen expression in different clones revealed that immunogenic and low metastatic variants expressed levels of H-2 antigens higher than the tumorigenic and metastatic clones. Finally, by using cyclophosphamide (Cy) treatment and adoptive transfer of immune spleen cells we were able to eradicate macroscopic 3LL pulmonary metastasis. These results demonstrate that the decrease of metastatic ability in u.v.-treated cells was mainly due to an increase in their immunogenicity and H-2 antigen expression.     

Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10⁵ copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3′ portion and polyomavirus sequence (to minimize homology with human genome) on the 5′ portion. The 3′ portion also consists of mismatch sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest normal K-ras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second–stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10⁵. This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles. Hum Mutat 10:322–325, 1997. © 1997 Wiley-Liss, Inc.     

Mitotic and post mitotic consequences of genomic instability induced by oncogenic Ha-Ras

Induced expression of a mutant human Ha-ras oncogene in NIH3T3 cells leads to the rapid production of multicentric chromosomes, acentric chromosome fragments, double minute chromosomes, increased heteroploidy, and increased capacity to undergo gene amplification. In this study we have used fluorescent-in-situ hybridization (FISH) to demonstrate that induction of the Ha-ras oncogene also leads to disruption of the mitotic machinery, resulting in aberrant mitoses and abnormal daughter cells. Cells induced to express an oncogenic Ha-ras transgene accumulate chromosomes that lag outside of the rest of the chromosomal architecture, chromosomes that form bridges between daughter nuclei at anaphase, and that form micronuclei. Many of these mitotic aberrations contain structurally abnormal chromosomes. Theseras-induced changes were suppressed by the introduction of a gene encoding the dominant negative effector ofras, raf 301. Expression ofraf301 in cells induced to express Ha-ras reduced the level of growth in soft agar, chromosome aberrations, mitotic aberrations, and frequency of gene amplification. These data provide evidence for an association between Ha-ras induced transformation, chromosome aberrations and gene amplification. Furthermore they offer insight into how the cell responds to the formation of aberrant chromosomes, and how disrupting chromosomal architecture could lead to further imbalances in the distribution of genetic material between daughter cells.     

“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical Cancer: A Study of 31 Cases

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for “low-risk” (types 6 and 11) and “high-risk” (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. “Low-risk” HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of “high-risk” human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that “high-risk” HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.     

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.     

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.     

Peritoneal metastatic model for human scirrhous gastric carcinoma in nude mice

We established a peritoneal-metastatic model for scirrhous gastric carcinoma. Peritoneal metastasis had developed after intraperitoneal inoculation of OCUM-2MD3 cells in nude mice. This cell line was derived from a peritoneal-metastatic nodule at the mesenterium after orthotopic implantation of OCUM-2M cells which developed no peritoneal metastasis after intraperitoneal inoculation. The histologic findings of orthotopic-implanted tumor in the stomach show scirrhous type while those of subcutaneous-implanted tumor show medullary type. There might be factors, in OCUM-2MD3 cells, which are responsible for peritoneal metastasis. We next investigated the differences in the biological behavior of the original OCUM-2M and the derived variant OCUM-2MD3. Morphology and growth activity of the two cell lines were similar to each other. The specific chromosomes, add(6)(g13), del(7)(q21.2) and inv(11)(pl3q21), were found in OCUM-2MD3 cells but not in OCUM-2M cells. While the oncogenes amplification by OCUM-2M cells was found in K-sam and c-myc, that by OCUM-2MD3 cells was found only in c-myc. The expression of E-cadherin by OCUM-2MD3 cells was decreased compared with that of OCUM-2M cells. Expression level of β1-integrin of OCUM-2MD3 cells was higher than that of OCUM-2M cells. The binding and invasion activity of OCUM-2MD3 cells were higher than those of OCUM-2M cells, and were decreased by anti-β1-integrin antibody. The invasion activity of OCUM-2MD3 cells was increased in the presence of peritoneal fibroblast. In this study, it was suggested that orthotopic implantation of cancer cells might have an effect on the acquisition of metastatic ability. β1-integrin and peritoneal fibroblasts might be correlated with peritoneal metastasis. This peritoneal-metastatic model should be useful for analysing the mechanism of peritoneal metastasis of human scirrhous gastric cancer.     

Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA)

From 10 to 30% of lung carcinomas examined to date contain mutant K-ras genes. We report here that the mutant-allele-specific amplification (MASA) method may be useful for detection of the K-ras mutations in cells obtained from the sputum of patients with lung cancer. The PCR product from one of five patients revealed an alteration when mixed oligonucleotides representing variants of the second letter at codon 12 of this gene were used as 5′ primers, and further experiments showed a mutation of GGT (Gly) to GAT (Asp)at codon 12. The MASA system could also be applied to an examination of metastatic lung carcinomas, particularly from adenocarcinomas in colon and pancreas in which frequent K-ras mutations are detected, and to mass-screening for colorectal tumors using DNA isolated from feces as template. © 1993 Wiley-Liss, Inc.