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.     

Analysis of KRAP expression and localization,and genes regulated by KRAP in a human colon cancer cell line

We previously identified the human KRAP (Ki-ras-induced actin-interacting protein) gene from the cDNA library of human colon cancer HCT116 cells as one of the genes whose expression levels were up-regulated by activated Ki-ras. Although the KRAP gene is structurally conserved from fish to mammalian species, the expression pattern and function of KRAP still remain to be elucidated. Here, we have generated a specific polyclonal antibody for KRAP and characterized the histological expression of KRAP in mouse tissues. KRAP was ubiquitously expressed in mouse tissues, with high levels in pancreas, liver, and brown adipose tissues, and KRAP was co-localized with filamentous actin along the apical membranes in both pancreas and liver tissues. A subfractionation study revealed that KRAP is a cytoplasmic protein and that the majority is associated with the cytoskeleton. Furthermore, microarray gene expression profile by inhibiting KRAP expression in HCT116 cells showed that several receptors and signal molecules frequently deregulated in cancers were differentially expressed in the KRAP-knockdown cells. All of these results suggested that KRAP might be a cytoskeleton-associated protein involving the structural integrity and/or signal transductions in human cancers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gastric adenoma — carcinoma sequence with special reference to p53 and Ki-ras gene alterations

With the aim of detecting the timing of p53 and Ki-ras gene alterations in the gastric adenoma-carcinoma sequence, 19 early gastric adenocarcinomas arising from adenomas were studied. Immunohistochemically, 5 adenocarcinomas were positive for p53; 3 focally and 2 diffusely. The p53 point mutations were detected in a focal area with p53 immunoreactivity in 2 of the 5 p53-positive adenocarcinomas. This indicated that p53 point mutations may play a less crucial part in malignant conversion of adenoma to adenocarcinoma in the stomach than in the colon. No Ki-ras gene mutations at codons 12 and 13 were detected in any lesion. These results suggest that the adenoma-carcinoma sequence in the stomach has a different mechanism from that in the colon.Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan     

Undifferentiated carcinoma of the pancreas: analysis of intermediate filament profile and Ki-ras mutations provides evidence of a ductal origin

Undifferentiated carcinomas and osteoclast-like giant cell tumours of the pancreas commonly contain foci of neoplastic ductal glands. To test the hypothesis that undifferentiated carcinomas and osteoclast-like giant cell tumours have a ductal origin, the immunocytochemical cytokeratin pattern and the frequency and type of Ki-ras mutations at colon 12 were studied in a series of 17 undifferentiated carcinomas and two osteoclast-like giant cell tumours. The cytokeratin features of undifferentiated carcinomas and osteoclast-like giant cell tumours were compared with those found in 10 ductal adenocarcinomas, 20 acinar cell carcinomas, 25 neuroendocrine tumours, and 15 solid-pseudopapillary tumours. All undifferentiated carcinomas and osteoclast-like giant cell tumours stained with at least one cytokeratin antibody, and 13/19 of them with antibodies against cytokeratins 7, 8, 18, and 19. The latter cytokeratins were expressed in all ductal adenocarcinomas, but only in 15/20 acinar cell carcinomas, 2/25 neuroendocrine tumours, and 1/15 solid-pseudopapillary tumours. In addition to cytokeratin, 15/19 undifferentiated carcinomas/osteoclast-like giant cell tumours were positive for vimentin. Ki-ras mutations at codon 12 were found in 10 undifferentiated carcinomas and one osteoclast-like giant cell tumour from which DNA could be successfully amplified. The Ki-ras mutation patterns were analysed in six tumours and corresponded to those typical of ductal adenocarcinomas. In tumours with ductal and anaplastic components, both components revealed identical mutation patterns. From these findings, it is concluded that both undifferentiated carcinomas and osteoclast-like giant cell tumours belong to the pancreatic tumours that show a ductal phenotype. Since undifferentiated carcinomas and osteoclast-like giant cell tumours share the same cytokeratin and Ki-ras features, they are probably derived from the same cell lineage. © 1998 John Wiley & Sons, Ltd.     

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.     

Expression of cyclin D1 and c-Ki-ras gene product in human epithelial ovarian tumors

The expression of cyclin Dl gene product in human ovarian tumors was studied. We found that cyclin D1 is expressed at high levels in several ovarian cancer cell lines. Immunohistochemical study also showed that a significant proportion of primary ovarian tumor tissues overexpressed cyclin D1 gene product. Clear nuclear staining of cyclin Dl protein was detected in 28% of the cases. We also characterized the expression of c-Ki-ras gene product in ovarian cancer cell lines and tumor tissues. Amplification or overexpression of this protooncogene has been reported in ovarian tumors from Taiwan. These results show that c-Ki-ras is strongly expressed in PA-1 and NIH: OVCAR-3 cells in which cyclin D1 also expressed at high levels. Specific cytoplasmic staining of c-Ki-ras protein was detected in 11 tumors (52%). Statistical analyses show a strong positive correlation between cyclin D1 and c-Ki-ras immunoexpression. Thus, these data support the ideas that cyclin D1 may be involved in the pathogenesis of ovarian cancer, and coactivation of cyclin D1 and c-Ki-ras gene expression may represent one of the major pathways that lead to the development of ovarian cancer in Taiwan.     

Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas,and association of codon-12 valine with early mortality

The point mutations occurring in codons 12 and 13 of Ki-ras in 78 patients with colorectal carcinoma (31 Dukes' A and B, 21 Dukes' C, and 26 Dukes' D) have been determined by allele-specific oligonucleotide hybridization and sequencing. Duplicate samples of invasive primary carcinoma, adjacent normal tissue, and available lymph node and liver metastases from the same patients were microdissected from paraffin sections. There were no differences in the mutation rate between primary carcinomas and secondary deposits: 26 of 78 (33 per cent) primary carcinomas, 10 of 32 (31 per cent) lymph node metastases, and 10 of 26 (38 per cent) liver metastases. Multiple sampling revealed frequent heterogeneity within carcinomas: 9 of 26 primaries with Ki-ras mutations also contained areas of carcinoma with only the wild-type gene, implying that Ki-ras mutation, even when present in a colonic carcinoma, may not have been necessary for establishing the malignant phenotype. Also, 2 of 26 (8 per cent) Dukes' D patients had a mutation in their primary carcinoma but none in liver metastases and 6 of 47 (13 per cent) Dukes' C and D patients had mutations in liver or lymph node metastases but none in the primary carcinoma. Such heterogeneity may modify the effectiveness of novel therapies targeting mutant Ki-ras function, such as farnesyltransferase inhibition. Mutation of codon 12 from GGT (glycine) to GTT (valine) was more prevalent in primary and metastatic deposits of Dukes' C/D carcinomas (P=0·01) than in primary carcinomas from Dukes' A/B patients. Mutations of codon 12 to GAT, AGT, GCT and codon 13 GGC to GAC were also found, but no correlation with carcinoma aggressiveness was apparent. Follow-up of 71/78 patients (up to 12 years) revealed decreased overall survival (P=0·001) in patients with the GGT to GTT transversion in codon 12, even when the analysis was restricted to Dukes' D cases, supporting the suggestion that this mutation may confer a more aggressive phenotype in colorectal carcinoma. © 1998 John Wiley & Sons, Ltd.     

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.     

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.     

p21 ras protein expression in benign and malignant

The ras oncogenes encode for GTP binding and GTPase active proteins of relative molecular mass 21 000 (p21^ras) which are involved in the transduction of stimuli for cell proliferation. There have been conflicting reports about the detection and significance of expression of p21^ras protein in human breast disease as determined by immunohistochemistry. The antibody Y13-259, which detects a single protein of M_r 21 000, has been applied immunohistochemically to frozen sections of normal, benign proliferative breast, fibroadenomas, and carcinomas. Uniform staining of normal breast epithelium and myoepithelium was found, with occasional stronger staining in areas of epithelial hyperplasia in benign breast disease. Contrary to previous reports, decreased expression, usually heterogeneous, was found in half of the carcinomas examined. Thirty per cent of the carcinomas exhibited heterogeneous staining stronger than that of normal breast, interpreted as increased expression of p21^ras protein. This did not relate to tumour grade or node status but showed a significant correlation with proliferation rate as determined by the monoclonal antibody Ki-67. This study confirms previous reports that p21^ras protein expression is a feature of normal cells, and has identified increased expression in 30 per cent of tumours associated with higher proliferation rates, which is a lower incidence than previously claimed when a different antibody was employed.     

POTENTIAL FALSE-POSITIVE RESULTS WITH ANTIGEN ENHANCEMENT FOR IMMUNOHISTOCHEMISTRY OF THE p53 GENE PRODUCT IN COLORECTAL NEOPLASMS

Aberrant crypt foci (ACF) are putative precursor lesions of colon cancer, recently identified on the methylene blue-stained mucosal surface of human colon. No mutations in K- ras or p53 genes were found by non-radioactive single-strand conformation polymorphism analysis in 14 ACF collected from five patients. Using the more sensitive method of allele-specific polymerase chain reaction (PCR) for K- ras , 8 of 14 ACF were found to contain K- ras mutations, suggesting that mutated cells are present in minute clones in ACF. No dysplasia was observed in any of the ACF containing a mutated clone. The presence of K- ras mutations in ACF suggests that these lesions occur at a very early stage in human colorectal carcinogenesis.     

Pancreatoblastoma in an adult: its separation from acinar cell carcinoma

Pancreatoblastomas are rare tumours, which usually occur in childhood. Here we describe a pancreatoblastoma in a 39-year-old woman. The tumour was located in the tail of the pancreas and consisted of cells forming well-differentiated acinar structures and scattered solid components (squamoid corpuscles). Immunocytochemically, the acinar components were positive for pancreatic enzymes and pancreatic stone protein, while the cells of the squamoid corpuscles lacked these markers. There was no p53 overexpression nor any mutation at codon 12 of the Ki-ras oncogene. The main differential diagnosis of this tumour was acinar cell carcinoma, because both tumours have a number of features in common (scattered solid components, positivity for pancreatic enzymes, lack of p53 overexpression and of Ki-ras mutation). Findings which distinguished the pancreatoblastoma and spearated it from acinar cell carcinoma were the negativity of the solid components (squamoid corpuscles) for neuroendocrine markers and their very weak keratin positivity. As the patient is alive and well 30 months after tumour resection, this pancreatoblastoma also differs in biology from the usual acinar cell carcinoma.     

K-ras AND p53 MUTATIONS IN HUMAN COLORECTAL ABERRANT CRYPT FOCI

Aberrant crypt foci (ACF) are putative precursor lesions of colon cancer, recently identified on the methylene blue-stained mucosal surface of human colon. No mutations in K- ras or p53 genes were found by non-radioactive single-strand conformation polymorphism analysis in 14 ACF collected from five patients. Using the more sensitive method of allele-specific polymerase chain reaction (PCR) for K- ras , 8 of 14 ACF were found to contain K- ras mutations, suggesting that mutated cells are present in minute clones in ACF. No dysplasia was observed in any of the ACF containing a mutated clone. The presence of K- ras mutations in ACF suggests that these lesions occur at a very early stage in human colorectal carcinogenesis.     

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.     

Frequent p53 gene mutations in serrated adenomas of the colorectum

Serrated adenoma has been recently proposed as a distinct histological lesion of the colorectum. This study examined p53 immunoreactivity, mutations of exons 5–8 of the p53 gene, codon 12 of the Ki-ras gene by PCR–SSCP analyses, and microsatellite instability in 19 serrated adenomas, ten adenocarcinomas in/with serrated adenomas, 23 hyperplastic nodules, four hyperplastic polyps and 29 tubular adenomas of the colorectum. Eleven of 11 (100 per cent) serrated adenomas had p53 immunoreactivity and all six (100 per cent) adenocacinomas in/with serrated adenomas exhibited moderate to severe p53 immunoreactivity. It was confirmed that 9 of 19 (47 per cent) serrated adenomas and 5 of 10 (50 per cent) adenocarcinomas in/with serrated adenomas harboured p53 gene mutations. On the other hand, no p53 gene mutation was detected in the other colorectal lesions. Meanwhile, 11 (58 per cent) serrated adenomas and six (60 per cent) adenocarcinomas in/with serrated adenomas had Ki-ras gene mutations, as also did 9 of 23 (39 per cent) hyperplastic nodules, 3 of 4 (75 per cent) hyperplastic polyps, and 12 of 29 (41 per cent) tubular adenomas. Microsatellite instability was detected in one (5 per cent) serrated adenoma and one (10 per cent) adenocarcinoma in a serrated adenoma. The other lesions did not show microsatellite instability. Serrated adenomas had significantly frequent p53 gene mutations compared with hyperplastic lesions or tubular adenomas (p<0·005). On the other hand, they did not exhibit significant differences in mutations of the Ki-ras gene or in microsatellite instability. Genetic changes were then examined in small parts of serrated adenomas, such as the upper or lower parts of crypts, to determine the extent of gene mutations by using a microdissection technique. Exon 15 of the APC gene and the DCC gene, in addition to the p53 and Ki-ras genes and microsatellite instability, were analysed. Identical mutations of the p53 gene were found in both invasive adenocarcinomas and adjacent serrated adenomas by direct sequencing, suggesting single clonal origins for those lesions. Mutations of the APC gene and microsatellite instability were heterogeneous in some lesions. No loss of heterozygosity (LOH) of the DCC gene was found. These findings suggest that mutations of the p53 gene are the most characteristic genetic alterations in serrated adenomas, as a relatively early event in a multistep carcinogenic pathway of this type of colorectal lesion, that might be distinct from the ordinary adenoma–carcinoma sequence or from carcinogenesis via mutations of mismatch repair genes. Copyright © 1998 John Wiley & Sons, Ltd.     

Transfection of activated ras into an excitable cell line (AtT-20) alters tetrodotoxin sensitivity of voltage-dependent sodium current

The sensitivity of voltage-dependent sodium current to the sodium channel blocker tetrodotoxin (TTX) is altered by transfection of a c-Ha-ras oncogene into an excitable cell line. Control AtT-20 cells, a cell line derived from a mouse anterior pituitary tumor, were found to express both a TTX-sensitive and a TTX-resistant sodium current. AtT-20 cells transfected with the c-Ha-ras gene expressed only a TTX-sensitive current. Properties of TTX-sensitive and -resistant currents were also examined. No differences in voltage dependence of activation or inactivation between the TTX-sensitive and -resistant currents were observed. The rate of inactivation of the TTX-resistant current in control cells was slower, than that of the TTX-sensitive current in either control or ras-transfected AtT-20 cells.     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

K- ras mutation may promote carcinogenesis of endometriosis leading to ovarian clear cell carcinoma

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.