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.

Diagnosis and monitoring of colorectal cancer by L6 blood serum polymerase chain reaction is superior to carcinoembryonic antigen-enzyme-linked immunosorbent assay.

PURPOSE: The aim of this study was to compare carcinoembryonic antigen levels with detection of messenger ribonucleic acid coding for the tumor-associated antigen L6 in patients with colorectal cancer. Not only are carcinoembryonic antigens expressed by the corresponding tumor cell, but the messenger ribonucleic acid of tumor-associated antigens, in contrast, is produced exclusively by viable tumor cells. METHODS: L6 messenger ribonucleic acid was determined by reverse-transcription polymerase chain reaction. Carcinoembryonic antigen was measured by the enzyme-linked immunosorbent assay technique, with a cutoff value of 40 microg/l. Blood serum was sampled from 187 patients with colorectal cancer. Statistical significance was calculated with the McNemar chi-squared test. RESULTS: Preoperatively, 79 percent of patients in all stages were positive for L6 messenger ribonucleic acid, whereas only 35 percent had elevated carcinoembryonic antigen titers (P < 0.001). In Dukes A tumors, 84.9 percent of patients were positive for L6 messenger ribonucleic acid, whereas carcinoembryonic antigen was elevated in only 16.9 percent of patients. Only in Dukes D tumors did the enzyme-linked immunosorbent assay for carcinoembryonic antigen exhibit the same sensitivity as reverse-transcription polymerase chain reaction for L6 messenger ribonucleic acid. Recurrence was detected significantly earlier by reverse-transcription polymerase chain reaction for L6 messenger ribonucleic acid than by enzyme-linked immunosorbent assay for carcinoembryonic antigen. CONCLUSION: L6 is more sensitive and precise than carcinoembryonic antigen in diagnosing and monitoring colorectal cancer.

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.

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.