Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas.
Ruggeri BA. Huang L. Wood M. Cheng JQ. Testa JR.
Department of Pathology and Laboratory Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA.
AKT2, an oncogene encoding a protein serine-threonine kinase implicated in phosphatidylinositol-3-OH kinase signaling, is amplified in some human ovarian and pancreatic carcinomas. We previously demonstrated that the tumorigenicity and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cell lines with amplified AKT2 could be markedly reduced by transfection with antisense AKT2 constructs. To evaluate further the extent of AKT2 alterations in PDAC, DNA and immunohistochemical analyses were performed to assess amplification or overexpression of AKT2, respectively, in 72 PDACs. Thirty-five PDACs were subjected to Southern analyses, and AKT2 amplification was detected in seven tumors (20%). Forty-one formalin-fixed PDAC specimens were examined immunohistochemically with an anti-AKT2 monoclonal antibody, and moderate to intense staining was observed in eight tumors (20%). AKT2 immunostaining paralleled AKT2 genomic status in each of four cases in which both Southern and immunohistochemical analyses were performed. No obvious relationship was observed between AKT2 status and tumor TNM stage or grade. These observations suggest the utility of immunohistochemical analysis in assessing alterations of AKT2 in human cancers. Furthermore, the role played by the AKT2 kinase in the signaling pathways of various mitogenic growth factors implicated in the development of pancreatic cancer suggests that alteration of AKT2 may be an important component in the pathogenesis of a substantial subset of PDACs.
Characterization of rare p53 mutants from carcinogen-treated albumin-simian virus 40 T-antigen transgenic rats.
Haas MJ. Pitot HC.
Department of Oncology, Medical School, University of Wisconsin, Madison 53706, USA.
The p53 gene has been either mutated or deleted in most human tumors examined to date. Mutations in the specific DNA-binding domain are the most common p53 mutations and are of interest because they may produce p53 molecules with transcriptional capabilities unlike those of the wild-type (WT) p53 protein. Mutations in the rat p53 gene were found in hepatic neoplasms of carcinogen-treated transgenic rats that express simian virus 40 (SV40) large T-antigen (TAg). Because this result was unexpected, we examined some of the biochemical and biological properties of the mutant proteins. Corresponding nucleotide changes were made by site-directed mutagenesis of the rat p53 cDNA, which was then inserted into a eukaryotic expression vector and transfected into the human hepatocyte cell line Hep 3B. Four of the mutant p53 molecules from rat hepatomas retained a strict WT conformation. Two others existed in both WT and mutant conformations. All of the mutant proteins were able to bind TAg as well as WT p53 did. Whereas the WT p53 protein was able to repress expression of a reporter gene containing a p53-response element (pSV2CAT), the missense-mutant p53 proteins induced transcription of the reporter to an extent equivalent to that of TAg. The mutant proteins also allowed TAg to induce the pSV2CAT reporter gene. The mutant molecules were able to enhance survival of Hep 3B cells, perhaps by preventing cell death, whereas expression of the WT p53 protein caused a reduction in cell number to nearly 10% of control levels. The results of these experiments suggest that the mutant p53 molecules observed in the carcinogen-treated transgenic rats may have unique properties that are important in carcinogenesis.
Few point mutations in elongation factor-1gamma gene in gastrointestinal carcinoma.
Frazier ML. Inamdar N. Alvula S. Wu E. Kim YH.
Department of Gastrointestinal Medical Oncology and Digestive Diseases, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Elongation factor-1 (EF-1) gamma is overexpressed in a high proportion of gastrointestinal cancers. The mechanism of overexpression has not been determined. The purpose of this study was to examine cDNA specimens from pancreatic and colorectal cancer for mutation in this gene, which would allow us to determine whether gene mutations are responsible for overexpression of EF-1gamma. In one colorectal carcinoma, we detected an A-->G transition at amino-acid codon 158 (T-->C in the sense strand) resulting in a change from a leucine to a serine. The base change was not detected in cDNA isolated from normal-appearing tissue from the same patient. We did not find mutations in another five colorectal carcinoma and five pancreatic cancer samples. Thus, although we detected a mutation in one tumor, the frequency of mutations was too low to account for the high frequency of overexpression of the EF-1gamma RNA in colorectal cancer. We also investigated other possible mechanisms of overexpression of the EF-1gamma RNA in this study. Slot-blot analysis of DNA isolated from colorectal cancers showed that the overexpression was not due to gene amplification. Using serum starvation to synchronize cultured cells, we showed that the overexpression was also not due to an increase in the number of cycling cells, as occurs in cancer. Using Southern blot analysis, we were unable to detect genome rearrangements that could have been responsible for the overexpression. In conclusion, the mechanism for overexpression of the EF-1gamma gene in colorectal and pancreatic cancer remains unknown. However, mutations in the coding sequence of the gene, gene amplification, and gene rearrangement do not account for the high frequency of overexpression of this gene, and the overexpression is not due to an increase in the number of cycling cells.
Inhibitory effect of matrilysin antisense oligonucleotides on human colon cancer cell invasion in vitro.
Momiyama N. Koshikawa N. Ishikawa T. Ichikawa Y. Hasegawa S. Nagashima Y. Mitsuhashi M. Miyazaki K. Shimada H.
Second Department of Surgery, Yokohama City University School of Medicine, Yokohama, Japan.
In colorectal cancer, matrilysin (matrix metalloproteinase-7) is mainly produced by the tumor cells themselves and is thought to play an important role in tumor invasion and metastasis. In the study reported here, we examined the effects of matrilysin antisense phosphorothioate oligonucleotides on both the expression of matrilysin and the invasive potential of the human colon cancer cell line CaR-1 in vitro. To select the most specific and potent oligonucleotide sequence, we performed extensive analyses of the binding specificities of all antisense candidates in the GenBank database by using a computer program we developed. As a result, a 15-mer matrilysin-specific antisense oligonucleotide that hybridizes to the coding region of matrilysin mRNA (AS-1) and a random control oligonucleotide (CL-1) were designed. Reverse transcription-polymerase chain reaction and western blot analysis demonstrated that 10 microM AS-1 suppressed matrilysin expression at both the mRNA level (92%) and protein level (64%). In vitro invasion assays demonstrated that this same concentration of AS-1 inhibited the ability of cells to invade a reconstituted basement membrane by 50% as compared with the ability of untreated cells to do so. On the other hand, CL-1, which had the same length and GC content as AS-1, did not show any inhibitory effect. These results demonstrate that the antisense oligonucleotide AS-1 inhibits matrilysin activities in a sequence-specific manner and suggest that AS-1 has the potential to be used as an anti-metastatic agent in an in vivo experimental model of colon cancer.