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.     

Genetic recombination in human melanoma and astrocytoma cell lines involves oncogenes and growth factor genes

We describe here the chromosomal distribution of sister chromatid exchanges (SCEs) in four human tumor cell lines (two melanomas and two astrocytomas), and have mapped the sister chromatid recombination (SCR) sites. A higher incidence of SCR sites than expected on the basis of chromosome length occurred in chromosomes 2, 4, 5 and 15 in both the RPMI 5966 and MEL57 melanoma cell lines, and in chromosomes 1, 5, 13 and 15 of the IJKt and GUVW astrocytoma cell lines. A majority of the recombination sites occurred close to chromosomal fragile sites. A third of these occurred at the same bands as fragile sites. The recombination sites involved the N-ras and the epidermal growth factor gene in the melanomas. In the astrocytomas, the N-ras, Rb and c-mos genes appeared to be involved in the recombination events. The β2-microglobulin gene was involved in both astrocytomas and one melanoma. The erb132 was involved in SCR only in the RPMI melanoma.     

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.     

Cytomorphological,cytogenetic, and molecular biological characterization of four new human renal carcinoma cell lines of the clear cell type

Four new permanent cell lines (RCC-A, -B,-C, and -D) derived from different human renal cell carcinomas of the clear cell type were established in tissue culture. The cell lines displayed characteristic differences in cell size and shape, which allowed individual identification by phase contrast microscopy. Ultrastructurally, the cell lines exhibited varying amounts of cytoplasmatic glycogen and lipid. Immunohistochemistry revealed co-expression of vimentin and cytokeratin in all cell lines. The mean population doubling time ranged from 27 h (RCC-A) to 104 h (RCC-D). RCC-B and -C cells produced slowly growing tumours after heterotransplantation into nude mice, whereas RCC-A and RCC-D cells were non-tumorigenic. The modal chromosome number was either near-diploid (RCC-A, -B, and -C) or near triploid (RCC-D). Clonal abnormalities affecting the short arm of chromosome 3 were seen in all cell lines. Northern blot analysis revealed no expression of the proto-on-cogenes c-fos, c-ros, and c-mos, whereas c-Ki-ras expression was observed in all cell lines. Expression of c-myc was observed in RCC-A, RCC-B, and RCC-D cells, whereas c-raf expression could be detected in RCC-B and RCC-D. Tumour suppressor gene p53 mRNA was observed in the cell line RCC-D.     

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction

Previous studies of c-myc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and elecrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher that than of small cell carcinoma (Wilcoxon rank sum test, Z=2.06, P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in non-small cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma). Received: 17 October 2000 / Accepted: 7 May 2001     

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.     

Co-amplification of 8p12 and 11q13 in breast cancers is not the result of a single genomic event

Epithelial cancers frequently have multiple amplifications, and particular amplicons tend to occur together. These co-amplifications have been suggested to result from amplification of pre-existing junctions between two chromosomes, that is, translocation junctions. We investigated this hypothesis for two amplifications frequent in breast cancer, at 8p12 and 11q13, which had been reported to be associated in Southern blot studies. We confirmed that both genomic amplification and expression of genes was correlated between the frequently-amplified regions of 8p and 11q, in array CGH and microarray expression data, supporting the importance of co-amplification. We examined by FISH the physical structure of co-amplifications that we had identified by array CGH, in five breast cancer cell lines (HCC1500, MDA-MB-134, MDA-MB-175, SUM44, and ZR-75-1), four breast tumors, and a pancreatic cancer cell line (SUIT2). We found a variety of arrangements: amplification of translocation junctions; entirely independent amplification of the two regions on separate chromosomes; and separate amplification of 8p and 11q sequences in distinct sites on the same rearranged chromosome. In this last arrangement, interphase nuclei often showed intermingling of FISH signals from 8p12 and 11q13, giving a false impression that the sequences were interdigitated. We conclude that co-amplification of the main 8p and 11q amplicons in breast tumors is not usually the result of a preceding translocation event but most likely reflects selection of clones that have amplified both loci. This article contains supplementary material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes

Amplification of oncogenes is an important mechanism that can cause gene overexpression and contributes to tumor development. The identification of amplified regions might have both prognostic and therapeutic significance. We used primary lung carcinomas and lung cancer cell lines for restriction landmark genomic scanning (RLGS) to identify novel amplified sequences. Enhanced RLGS fragments that indicate gene amplification were observed in primary tumors and lung cancer cell lines of both non-small cell lung cancer and small cell lung cancer. We identified one novel amplicon on chromosome 11q22, in addition to previously reported amplicons that include oncogenes MYCC, MYCL1 and previously identified amplification of chromosomal regions 6q21 and 3q26-27. Amplification of 11q22 has been reported in other types of cancer and was refined to an approximately 1.19 Mbp region for which the complete sequence is available. Based on a patient sample with a small region of low-level amplification we were able to further narrow this region to 0.92 Mbp. Genes localized in this region include two inhibitors of apoptosis (cIAP1 and cIAP2). Immunohistochemistry and western blot analysis identified cIAP1 and cIAP2 as potential oncogenes in this region as both are overexpressed in multiple lung cancers with or without higher copy numbers.     

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     

Visualization of INT2 and HST1 Amplification in oral squamous cell carcinomas

Oral squamous cell carcinoma (OSCC) develops along a multistep genetic pathway including loss of tumor suppressor genes and alteration of oncogenes. We characterized seven OSCC cell lines by classical and molecular cytogenetic analysis and fresh tumor and adjacent oral mucosa corresponding to three of the cell lines by molecular cytogenetics. We observed homogeneously staining regions (hsrs) in four of the seven cell lines, at 11q13 in three and at 11q23 and in an unidentified marker chromosome in the fourth. Amplification of band 11q13 occurs in 30–60% of head and neck squamous cell carcinomas. To determine whether INT2 and HST1, both located in band 11q13, are amplified in the tissues and cell lines and to confirm the chromosomal location(s) of the amplification, we used dual-color fluorescence in situ hybridization (FISH) with DNA probes for these genes and the chromosome 11 centromere. We report chromosomal localization of INT2/HST1 amplification in OSCC. Coamplification of INT2 and HST1 was detected in the hsrs in cultured tumor cells from the four hsr-containing tumors and in directly harvested tumor cells, which were available from only two of these tumors. Amplification was not present in tumors lacking hsrs or adjacent oral mucosa corresponding to any of the seven tumors. The observation of amplification in fresh tumor cells suggests that the amplification was present in the patients, may play a key role in the development and/or progression of OSCC, and is not due to karyotypic evolution in vitro. The absence of amplification in the adjacent mucosa suggests that 11q13 amplification is a relatively late event in OSCC tumorigenesis.     

The role of acentric chromosome fragments in gene amplification

We assessed the role of acentric chromosome fragments in gene amplification by using cell fusion techniques to introduce the fragmented chromosomes of a donor Chinese hamster ovary (CHO) cell line that contained the dihydrofolate reductase (dhfr)gene(s) into a CHO cell line deficient for dhfr.Chromosome fragments were successfully integrated into cells at a frequency of approximately 3%. Methotrexate-resistant variants arose much more frequently in two cell lines derived from these successful cell fusions than in wild-type CHO cells. The hybrid cell lines also amplified their dhfrgenes more readily than did the CHO cell line used as dhfrdonor.     

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.     

Amplification units and translocation at chromosome 17q and c-erbB-2 overexpression in the pathogenesis of breast cancer

 Hyperplasia without and with atypia is considered to be a precursor lesion for certain breast carcinomas. The cytogenetic events and the molecular pathology involved in the multistep process from normal to invasive carcinoma are unknown. To characterise the sequence of early genetic abnormalities of chromosome 17q and their biological consequences in the pathogenesis of breast cancer, we performed immunohistochemistry on 451 breast tissues including 180 normal breast specimens, 28 hyperplastic lesions without atypia and 44 with atypia, 100 cases of ductal carcinoma in situ (DCIS) and 99 cases of invasive ductal carcinoma. We correlated the overexpression of the c-ErbB-2 protein, the histological and the recently proposed differentiation classification of DCIS with the extent of DCIS. For fluorescence in situ hybridisation (FISH) analysis, different probes spanning the 17q region including the c-erbB-2 gene locus and those which are found adjacent, were used. Reverse painting and comparative genomic hybridisation (CGH) were performed on several breast cancer cell lines. c-ErbB-2 overexpression was observed in only 29% of DCIS and 23% of invasive carcinomas, but not in hyperplastic and normal tissue. c-ErbB-2 overexpression is correlated with poor differentiation in DCIS but not in invasive carcinoma. In DCIS, there was no correlation with the histological subtype classification. The average extent of DCIS is significantly increased from 13.81 mm in c-ErbB-2 negative cases to 29.37 mm in c-ErbB-2 positive cases. The increase was considered to be a possible consequence of the overexpression and is probably due to the previously described motility enhancing effect of the c-ErbB-2 protein. The histological and differentiation classification of DCIS did not correlate with the extent of disease. Using FISH, amplified genes at 17q12, always including the c-erbB-2 gene, were detected in all cases of DCIS and invasive carcinoma with c-ErbB-2 overexpression. The centromeric region and the NF1 locus, which is located between the centromere and c-erbB-2, were not amplified in any of the DCIS and invasive breast carcinomas, but co-amplification of the myeloperoxidase gene was detected in 3/5 DCIS and 1/5 invasive carcinomas with c-ErbB-2 overexpression. In contrast to c-erbB-2, immunohistochemical overexpression of their respective gene products was not observed. FISH, reverse painting and CGH show similar amplified genes with amplified c-erbB-2 in c-ErbB-2 overexpressing SK-BR-3 and BT474 human breast cancer cells. The amplified genes are part of two different amplicons. Extensive modifications of the 17q chromosomal region, caused by translocation, were also observed in these cell lines. It is concluded that the modifications of chromosome 17q, inducing overexpression of c-ErbB-2 protein, occur at the level of transition from hyperplasia to DCIS. They are preserved in invasive carcinoma with overexpression of c-ErbB-2 protein. This had led to the hypothesis that these modifications at 17q may lead to a larger extent of DCIS. Received: 23 July 1996 / Accepted: 9 December 1996     

Unique chromosomal location of amplified EGF receptor genes in EGF receptor-hyperproducing tumor cell line NA

The epidermal growth factor receptor (EGFR) gene was analyzed by in situ hybridization using a squamous cell carcinoma line NA, which has high numbers of EGF receptors and carries a 20-fold amplification of EGFR genes. NA cells are pseudotriploid (mode of chromosome number is 69) and have three copies of an apparently normal chromosome 7 together with several aberrant chromosomes. Strong hybridization signals were observed in the abnormal banding region of one of the aberrant chromosomes, MH1, which has no structural homology to chromosome 7. This MH1 chromosome was lost in NA-derived variant lines that possess reduced numbers of EGF receptors. These results are in contrast to previous findings that EGFR gene amplification is associated with structural alterations of the short arm of chromosome 7 and provide new evidence in regard to the location of the amplified EGFR gene in tumor cells.     

High resolution analysis of DNA copy-number aberrations of chromosomes 8, 13, and 20 in gastric cancers

DNA copy-number gains of chromosomes 8q, 13q, and 20q are frequently observed in gastric cancers. Moreover gain of chromosome 20q has been associated with lymph node metastasis. The aim of this study was to correlate DNA copy-number changes of individual genes on chromosomes 8q, 13q, and 20q in gastric adenocarcinomas to clinicopathological data. DNA isolated from 63 formalin-fixed and paraffin-embedded gastric adenocarcinoma tissue samples was analyzed by whole-genome microarray comparative genomic hybridization and by multiplex ligation-dependent probe amplification (MLPA), targeting 58 individual genes on chromosomes 8, 13, and 20. Using array comparative genomic hybridization, gains on 8q, 13q, and 20q were observed in 49 (77.8%), 25 (39.7%), and 49 (77.8%) gastric adenocarcinomas, respectively. Gain of chromosome 20q was significantly correlated with lymph node metastases (p = 0.05) and histological type (p = 0.02). MLPA revealed several genes to be frequently gained in DNA copy number. The oncogene c-myc on 8q was gained in 73% of the cancers, while FOXO1A and ATP7B on 13q were both gained in 28.6% of the cases. Multiple genes on chromosome 20q showed gains in more than 60% of the cancers. DNA copy-number gains of TNFRSF6B (20q13.3) and ZNF217 (20q13.2) were significantly associated with lymph node metastasis (p = 0.02) and histological type (p = 0.02), respectively. In summary, gains of chromosomes 8q, 13q, and 20q in gastric adenocarcinomas harbor DNA copy-number gains of known and putative oncogenes. ZNF217 and TNFRSF6B are associated with important clinicopathological variables, including lymph node status.     

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.