Correction to: The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.

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Stage-specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis.

To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions.

Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors.

BACKGROUND & AIMS: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples. METHODS: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay). RESULTS: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal. CONCLUSIONS: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

PURPOSE: The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. METHODS: Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. RESULTS: We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. CONCLUSION: Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Detection of mutations in the cDNA of the proliferation marker Ki-67 protein in four tumor cell lines.

The Ki-67 protein has an essential role in cell proliferation. It is present in all dividing cells of normal and tumor tissues, but absent in resting cells. At present, no data are available about any alterations in the gene of this protein that could contribute to its altered structure and function, resulting in tumor development. We therefore searched for mutations in the Ki-67 gene (MKI67). cDNAs from four tumor cell lines derived from carcinoma of the cervix (HeLa), colon (CXF94, SW480), and lung (A549) were prepared. Defined parts of the cDNA were amplified by specific primers, cloned into pCRII-Blunt-TOPO vector, and replicated in Escherichia coli. The sequence of the amplified products were determined by automated fluorescence sequencing. Eight different mutations were characterized in the four cell lines tested. One is a deletion of a single base at position 1496 causing a truncated protein, the second is a A433T exchange is a silent mutation, and the remaining six mutations result in an amino acid change that might alter the conformation of the protein. Our results show that several mutations exist within the Ki-67 protein's cDNA in four tumor cell lines. These mutations might provide a genetic basis for tumor development.

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions.

Effect of cyclooxygenase-2 antisense oligodeoxyribonucleotides in A-549 lung cancer cells.

BACKGROUND: Cyclooxygenase (COX)-2 is overexpressed in several tumor entities and seems to play a key role in carcinogenesis. This makes it a potential target in cancer therapy. MATERIALS AND METHODS: Twelve phosphorothioate-modified antisense oligonucleotides (asODNs) against six targets of COX-2 mRNA were transfected to A-549 lung carcinoma cells. COX-2 mRNA and protein levels were determined by quantitative RT-PCR and flow cytometry, respectively. Cell growth was assessed by measuring Alamar Blue reduction. RESULTS: The tested asODNs exhibited a range of activities. The most potent asODN reduced uninduced COX-2 mRNA to 66% and protein level to 75%, respectively. While this asODN did not influence cell growth, a 15% growth reduction was observed after transfection of another asODN which suppressed COX-2 mRNA to 71% and protein level to 84%. CONCLUSION: The use of asODNs directed against COX-2 mRNA is a promising approach to inhibit COX-2 expression in tumor cells.

Local generation of C-reactive protein in diseased coronary artery venous bypass grafts and normal vascular tissue.

BACKGROUND: Venous coronary artery bypass grafts (CABGs) are prone to accelerated atherosclerosis. In atherosclerotic diseases, serum C-reactive protein (CRP) levels have become an important diagnostic and prognostic marker. The origin of CRP in this setting remains to be elucidated. METHODS AND RESULTS: Monoclonal anti-CRP identified CRP expression in medial and intimal alpha-actin-positive smooth muscle cells (SMCs) of diseased CABGs with type V and VI lesions and also of native saphenous veins of atherosclerotic individuals. In addition, patent coronary arteries with type IV and V but not with type I through III lesions exhibited intense SMC staining for CRP. Calcified desobliterates of occluded coronary arteries with end-stage disease did not show SMC staining for CRP and were consistently negative for CRP mRNA, as detected by means of real-time polymerase chain reaction. However, CRP mRNA was expressed in 11 of 15 diseased CABGs and also in 10 of 15 native veins. By contrast, only 3 of 18 internal mammary and 4 of 12 radial arteries with virtually no atherosclerosis were positive for CRP mRNA. CONCLUSIONS: CRP is produced by SMCs of atherosclerotic lesions with active disease but not in end-stage plaques. The role of CRP constitutively expressed by normal vascular tissue in vein graft disease has yet to be elucidated.

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.

The Ki-67 antigen, pKi-67, is one of the most commonly used markers of proliferating cells. The protein can only be detected in dividing cells (G(1)-, S-, G(2)-, and M-phase) but not in quiescent cells (G(0)). The standard antibody to detect pKi-67 is MIB-1, which detects the so-called 'Ki-67 motif' FKELF in 9 of the protein's 16 tandem repeats. To investigate the function of these repeats we expressed three of them in an inducible gene expression system in HeLa cells. Surprisingly, addition of a nuclear localization sequence led to a complete absence of signal in the nuclei of MIB-1-stained cells. At the same time antibodies directed against different epitopes of pKi-67 did not fail to detect the protein. We conclude that the overexpression of the 'Ki-67 motif', which is present in the repeats, can lead to inability of MIB-1 to detect its antigen as demonstrated in adenocarcinoma tissue samples. Thereafter, in order to prevent the underestimation of Ki-67 proliferation indices in MIB-1-labeled preparations, additional antibodies (for example, MIB-21) should be used. Additionally, we could show in a mammalian two-hybrid assay that recombinant pKi-67 repeats are capable of self-associating with endogenous pKi-67. Speculating that the tandem repeats are intimately involved in its protein-protein interactions, this offers new insights in how access to these repeats is regulated by pKi-67 itself.

Proliferation marker pKi-67 affects the cell cycle in a self-regulated manner.

The proliferation marker pKi-67 is commonly used in research and pathology to detect proliferating cells. In a previous work, we found the protein to be associated with regulators of the cell cycle, controlling S-phase progression, as well as entry into and exit from mitosis. Here we investigate whether pKi-67 has a regulative effect on the cell cycle itself. For that purpose we cloned four fragments of pKi-67, together representing nearly the whole protein, and an N-terminal pKi-67 antisense oligonucleotide into a tetracycline inducible gene expression system. The sense fragments were C-terminally modified by addition of either a nuclear localization sequence (NLS) or a STOP codon to address the impact of their intracellular distribution. FACS based cell cycle analysis revealed that expression of nearly all pKi-67 domains and the antisense oligonucleotide led to a decreased amount of cells in S-phase and an increased number of cells in G(2)/M- and G(1)-phase. Subsequent analysis of the endogenous pKi-67 mRNA and protein levels revealed that the constructs with the most significant impact on the cell cycle were able to silence pKi-67 transcription as well. We conclude from the data that pKi-67 influences progression of S-phase and mitosis in a self-regulated manner and, therefore, effects the cell cycle checkpoints within both phases. Furthermore, we found pKi-67 mediates an anti-apoptotic effect on the cell and we verified that this marker, although it is a potential ribosomal catalyst, is not expressed in differentiated tissues with a high transcriptional activity.

VEGF isoforms and mutations in human colorectal cancer.

We wished to demonstrate vascular endothelial growth factor (VEGF) transcript polymorphism in human colon cancer. RNA was extracted from 25 primary human colorectal adenocarcinomas followed by VEGF transcript amplification, fragment elution, subcloning, positive selection via insert analysis and sequencing. Four distinct splice variants were consistently expressed in cancer, including VEGF121, VEGF165, VEGF189 and the newly identified truncated splice variant VEGF145. Six novel mutations were characterized, all of which occurred within the conserved expression site of the gene and which consequently were present in all splice forms. Five cancers exhibited single nucleotide changes and 1 cancer a 2-nucleotide deletion. A silent mutation was observed in exon 1 at position +70 relative to the amplification start site, a 1- and 2-base deletion with frameshift and protein truncation in exon 3 at positions +172 and +171/172, respectively, a transition mutation in exon 3 at position +248 and 2 transition mutations in exon 4 at positions +398 and +403. All of these sense mutations should alter protein conformation. To our knowledge, this is the first report of VEGF145 in solid malignancy. Its biologic activity remains to be determined. We have demonstrated a variety of sporadic mutations within human colorectal cancer VEGF mRNA. Mutant angiogenic VEGF may provide a genomic basis for the diversity of tumor-host response and may prove to be important in antisense oligonucleotide targeting, since all the different VEGF isoforms would have to be neutralized to prevent angiogenesis.

Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease.

OBJECTIVE: Various antimicrobial peptides such as defensins are part of innate immunity and contribute to the intestinal barrier that may be defective in inflammatory bowel disease (IBD). This study investigated beta-defensin mRNA and peptide expression in the colon from controls and patients with Crohn's disease, ulcerative colitis or unspecific colitis as inflammatory controls. METHODS: Mucosal mRNA expression was measured by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) with primers for human beta-defensin 1 (HBD-1) and human beta-defensin 2 (HBD-2) in CaCo-2 cells and in biopsies from 103 patients (33 controls, 24 Crohn's disease patients, 36 ulcerative colitis patients, 10 unspecific colitis patients). Paraffin-embedded tissue from colonic resections was tested for HBD-1 and HBD-2 peptides by immunohistochemistry. RESULTS: HBD-1 mRNA was expressed constitutively whereas HBD-2 was induced by pro-inflammatory cytokines in CaCo-2 cells. HBD-1 mRNA was detectable in 61% of control and Crohn's disease biopsies and 53% of ulcerative colitis biopsies. HBD-2 transcript was expressed differentially, with 18% of control biopsies positive as opposed to 34% in Crohn's disease and 53% in ulcerative colitis. HBD-2 mRNA but not HBD-1 mRNA was expressed preferentially in inflamed areas. Immunohistochemical investigation demonstrated the presence of defensin peptides in colonic epithelium as well as the differential induction in IBD. CONCLUSIONS: HBD-1 is expressed constitutively in colonic tissue irrespective of inflammation. HBD-2 is barely present in uninflamed colon but it is induced in inflammation. The lower expression of HBD-2 in Crohn's disease compared with ulcerative colitis indicates different responses of the mucosal innate defence. Defensins may play a crucial role in controlling pathogen invasion in IBD, although the functional significance remains to be established.