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Identification of a new tumor suppressor gene located at chromosome 8p21.3-22¹

STEFAN SEIBOLD^*,,2, CLAUDIA RUDROFF, MANFRED WEBER^*, JAN GALLE, CHRISTOPH WANNER and MARTIN MARX^*

^* Department of Internal Medicine I, Cologne General Hospital, Merheim Medical Center, Medical Faculty University of Cologne, Cologne, Germany;
Department of Surgery, University of Witten-Herdecke, Wuppertal, Germany; and
Department of Medicine, University Medical Center, Wuerzburg, Germany

²Correspondence: Department of Medicine, University Medical Center, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany. E-mail: seibold_s{at}medizin.uni-wuerzburg.de

SPECIFIC AIMS

Carcinogenesis is a multistage process resulting in the loss of proliferative control and a failure to undergo cellular differentiation due to progressive acquisition of genetic alterations activating proto-oncogenes or inactivating tumor suppressor genes. Aim of our study was to identify new genes involved in regulation of carcinogenesis by investigating the gene expression during induction of cellular differentiation and quiescence in a 3-dimensional (3D) cell culture model.

PRINCIPAL FINDINGS

1. Identification of a gene up-regulated during cellular transition from proliferation to quiescence and differentiation
We compared gene expression patterns of differentiated and quiescent human umbilical vein endothelial cells (HUVEC) in 3D collagen type I cell culture vs. undifferentiated and proliferating HUVEC in a conventional 2-dimensional (2D) culture using the technique of differential display RT-PCR. Thereby a 211 bp band up-regulated during cellular transition from proliferation to quiescence and differentiation was identified. Northern blot analysis confirmed about threefold up-regulation, reaching a maximum within 24 h and returning to baseline expression after 96 h of 3D cell culture compared with the expression in 2D culture.

2. Cloning and sequence analysis of MTSG1
Proceeding from the 211 bp sequence obtained from the differentially displayed band, a comparison with the GenBank® database revealed no homology to any known gene at the time of investigation. We therefore amplified the entire corresponding cDNA using the 5'- and 3'-rapid amplification of cDNA ends (RACE) protocol. The resulting 4.1 kb cDNA sequence was transmitted to the GenBank® database (accession #AF121259). According to the functional data, we named this gene mitochondrial tumor suppressor gene 1 (MTSG1). Meanwhile several GenBank® entries confirm our sequence (mainly est sequences) that remained unpublished beside the GenBank® database. Homology also exists to an partial cDNA sequence, previously published together with 100 unrelated cDNAs (GenBank® accession #AB033114). The MTSG1 cDNA contains an open reading frame of 1303 bp encoding a protein of 436 amino acids with a calculated molecular mass of 50 kDa. The amino acid sequence of MTSG1 contains two N-glycosylation sites, several cAMP, protein kinase C, casein kinase II, and tyrosine kinase phosphorylation sites, an N-myristoylation site, and two leucine zipper motifs. Secondary structure analysis predicts five coiled-coil motifs spanning almost 50% of the protein sequence, bordered by noncoiled amino- and carboxyl-terminal ends and interrupted by four small noncoiled regions. SMART, a simple modular architecture research tool, identified a chromosomal segregation ATPase domain in the carboxyl-terminal part of the MTSG1 protein sequence. PSORT, a computer program predicting nuclearly encoded mitochondrial proteins, identified a mitochondrial targeting sequence within the amino-terminal end.

3. Subcellular localization of MTSG1
Immunocytochemistry of recombinantly expressed MTSG1 demonstrated colocalization with mitochondria in human MIA PaCa-2 cells (Fig. 1 ). Subcellular fractioning confirmed detection of recombinant MTSG1 protein in the mitochondrial fraction. Molecular mass of the FLAG-tagged recombinant protein was 48 kDa and therefore slightly lower as predicted from the amino acid sequence, supporting the hypothesis of amino-terminal cleavage during mitochondrial import.

View larger version (9K): [in this window] [in a new window]   Figure 1. Confocal laser microscopy of recombinantly expressed FLAG-tagged MTSG1 in human pancreatic tumor cells (MIA PaCa-2). A) Recombinant MTSG1/FLAG as green fluorescence detected with a polyclonal rabbit anti FLAG M2 antibody (Sigma). B) Mitochondria as red fluorescence of the same cells detected with 100 nM Mitotracker Deep Red (Molecular Probes). The red fluorescence at the right and left bottom belongs to an untransfected cell. C) Combined fluorescence of panels A, B. The yellow signal demonstrates colocalization of MTSG1/FLAG and mitochondria.

4. Genomic structure of MTSG1
Database searches revealed that the human MTSG1 gene is located within a 1.2 megabase region at chromosome 8p21.3–22, commonly deleted in various tumors (accession no. of the genomic clone: AB020864). Two markers, D8S254 and AFM333THI, 0.89 and 0.26 Mb apart from MTSG1, confine the gene. Comparison between the full-length cDNA and the genomic sequence showed that the gene consists of 10 exons, spanning almost 55 kb of genomic DNA.

5. Tissue distribution of MTSG1 mRNA
Expression of MTSG1 mRNA was present in all tissues tested. We found high expression in pancreas, heart, brain, and placenta, moderate expression in lung, liver, and kidney, and low expression in skeletal muscle.

6. MTSG1 mRNA expression in pancreatic tumor cell lines and tumors
MTSG1 mRNA expression was not detectable in the undifferentiated (G3) and fast proliferating (doubling time 30 h) pancreas carcinoma cell line MIA PaCa-2 (Fig. 2 ). Moderately differentiated and fast proliferating pancreas carcinoma cell lines DAN G (G2, doubling time 33 h) and PATU 8902 (G2, doubling time 40 h) expressed moderate, but much lower levels of MTSG1 mRNA than normal pancreatic tissue (Fig. 2) . High expression of MTSG1 mRNA as observed from normal pancreatic tissue was seen in the moderately differentiated, but slowly proliferating pancreas carcinoma cell line PATU 8988S (G2, doubling time 60 h), highly differentiated and slowly proliferating cell lines Capan 1 (G1, doubling time 50–100 h), and Capan 2 (G1, doubling time 96 h) (Fig. 2) . To circumvent in vitro effects of cell culture we also compared MTSG1 mRNA levels in an undifferentiated pancreas tumor (G3) expressing almost none MTSG1 mRNA (Fig. 2) . Inflammation within the tumor as an unspecific factor responsible for down-regulation of MTSG1 mRNA was excluded by demonstration of high expression of MTSG1 mRNA in tissue from a patient with pancreatitis (Fig. 2) .

View larger version (25K): [in this window] [in a new window]   Figure 2. MTSG1 mRNA expression in different tumor cell lines and tissues. MTSG1 mRNA expression is not detectable in the undifferentiated and fast proliferating pancreas carcinoma cell line MIA PaCa-2 and in tissue from an undifferentiated pancreatic tumor. In comparison, the slowly proliferating pancreas cell lines Capan 1, Capan 2, and PATU8988S as well as normal pancreatic tissue and tissue from patients with pancreatitis express high levels of MTSG1 mRNA. Intermediately proliferating pancreas carcinoma cell lines DAN G and PATU 8902 expressed intermediate levels of MTSG1 mRNA. The breast carcinoma cell line MCF 7 expresses MTSG1 mRNA as well. ß-Actin was used as an internal control to ensure equal template RNA. PCR products were electrophoresed on 1.5% agarose gels. Experiments were repeated 3 times and the gel shown was representative for all experiments.

7. Proliferation assay of MIA PaCa-2 cells with recombinantly expressed MTSG1
The pancreatic tumor cell line MIA PaCa-2 itself does not express MTSG1 mRNA (Fig. 2) . Therefore, the functional role of MTSG1 on the proliferation rate of tumor cells was investigated by analyzing BrdU incorporation into cellular DNA of MIA PaCa-2 cells after recombinant expression of MTSG1. A 30% ± 6.9% (given as mean±SE) reduction in BrdU incorporation was demonstrated in MIA PaCa-2 cells transfected with MTSG1 vs. MIA PaCa-2 cells transfected with a control vector, implying a regulation of cellular proliferation by MTSG1.

CONCLUSIONS AND SIGNIFICANCE

Here we report the identification and characterization of a tumor suppressor gene named MTSG1, located near marker D8S254 at chromosome 8p21.3–22. In many previous studies this chromosomal locus was predicted to hold such a tumor suppressor gene in breast cancer, esophageal cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, prostate cancer, urinary bladder carcinoma, and head and neck squamous cell carcinoma. Functional evidence for tumor suppressor genes at this chromosomal locus was further confirmed by previous studies showing a reduced tumorigenicity and metastatic potential of colorectal and prostate cancer cells after monochromosome 8 transfer. We identified MTSG1 < 0.9 Mb apart from the D8S254 marker locus with potential tumor suppressor activity by repressing tumor cell proliferation. We discovered MTSG1 by investigating the molecular regulation of cellular transition from proliferation to quiescence and differentiation in a 3D cell culture model. MTSG1 expression was thereby transiently up-regulated during initiation of the differentiated and quiescent cellular phenotype in 3D culture. The discrepancy of transient up-regulation of MTSG1, despite permanent cellular quiescence and differentiation in the 3D cell culture model, is not contradictory to a regulation of cellular proliferation by MTSG1. In fact, this phenomenon is well known in other regulators of cellular proliferation like the tumor suppressor gene p53. Maintenance of cellular quiescence despite declining p53 levels is thereby subsequently controlled by other regulators of cellular proliferation, such as pRb and p27CDKI. Further support for the hypothesis of a tumor suppressor function of MTSG1 arises from studies investigating its mRNA expression in different pancreatic tumors and tumor cell lines. These studies approved a reverse correlation of MTSG1 mRNA expression with cellular proliferation and differentiation, showing low expression in undifferentiated proliferating tumor cells and high expression in differentiated and slowly proliferating tumor cells. High expression found in tissue from pancreatitis excludes down-regulation of MTSG1 mRNA in tumor samples as a result of tumor inflammation. Further support for the hypothesis that MTSG1 regulates cellular proliferation arises from studies in MIAPaCa-2 cells expressing no native MTSG1 mRNA, where we recombinantly expressed MTSG1. An inhibitory effect of MTSG1 on cellular proliferation was demonstrated in these experiments by a 30% reduction in BrdU uptake in MTSG1 transfected cells compared with control transfected cells.

MTSG1 encodes a precursor protein of 436 amino acids imported into mitochondria as shown by Western blot analysis and immunohistochemistry. Mitochondrial localization of nuclearly encoded proteins is a common finding since 95% of mitochondrial proteins are nuclearly encoded and subsequently imported into mitochondria. Regulation of cellular proliferation by mitochondrial proteins may result from different mitochondrial functions. As the major source of energy supply, mitochondria influence cellular proliferation at energetic checkpoints at G1-S and G2M boundaries to ensure that cellular ATP content is above the critical threshold required for successful passage through that cell cycle phase. Mitochondria also contribute to the regulation of cellular proliferation through the production of reactive oxygen intermediates (ROI). ROI have been previously implicated as messengers in intracellular signal transduction mechanisms regulating transcription factors (AP-1, NF-B), kinases of the mitogenic signaling pathways (MAPK, BMK-1) and other cell cycle regulators, such as p53. MTSG1 could regulate mitochondrial functions by interaction with other mitochondrial proteins through the coiled-coil secondary structure containing two leucine zipper motives. Similar secondary structures are found in other regulatory proteins, like in the enhancer binding protein (C/EBP), the Jun/AP1 family of transcription factors, the fos-oncogene, the fos-related proteins fra-1 and fos B, and in the C-myc, L-myc, and N-myc oncogenes. A chromosome segregation ATPase domain was identified from the conserved domain database within the TSG1 protein sequence. However, additional domains not present in the MTSG1 protein are required for a DNA segregation activity, implying that MTSG1 is not involved in mitochondrial DNA segregation.

In conclusion we identified a tumor suppressor gene near marker D8S254 at chromosome 8p21.3–22, where existence of a tumor suppressor has been proposed for a long time. Tumor suppression is controlled by regulation of cellular proliferation via alteration of mitochondrial processes.

View larger version (57K): [in this window] [in a new window]   Figure 3. Schematic summary assuming the function of MTSG1. MTSG1 is a nuclear gene encoding a preprotein, finally imported into mitochondria. Transient up-regulation of MTSG1 results in a retroactive effect of mitochondria on the nucleus initiating cellular quiescence. Down-regulation of MTSG1 activates the cell cycle and results in tumor progression.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0934fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0934fje

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